Methods for liquid direct fluorescent antibody intracellular virus detection

ABSTRACT

The present invention describes a liquid direct fluorescence antibody assay that is rapid and sensitive to detect respiratory virus in infected cells. The assay includes centrifugation of the specimen, incubation of sample and reagents in solution, and detection of the absence or presence of respiratory virus. Sapogenin is used as a detergent to permeabilize the cells for entry of the monoclonal antibodies to react with intracellular antigens. The cells are stained with fluorescently labeled monoclonal antibodies against the viral antigens along with a background stain and a fluorescent nuclear stain. This counter staining decreases background and allows co-localization of antigen and nuclear structures for enhanced detection.

FIELD OF INVENTION

This invention is related to processing biological samples for directvirus detection in a liquid format. For example, the sample may bederived from the respiratory system. The detection method may useantibodies that directly bind to a viral antigen thereby allowingidentification as well as detection. In some instances, the antibodiesare labeled monoclonal antibodies. The method may be integrated with adevice comprising an algorithm capable of differentiating between aplurality of fluorescent signals.

BACKGROUND

Virus infections (i.e, for exmaple, influenza A and B viruses) areresponsible for yearly epidemics in both children and adults. Illnessescaused by influenza A and B viruses are clinically indistinguishable andmay cocirculate Van Voris et al., “Influenza viruses” p. 267-297. In: R.B. Belshe (ed.), Textbook Of Human Virology. PSG Publishing Co.,Littleton, Mass. (1984). Antiviral chemoprophylaxis and therapy iscurrently very limited (i.e., for example, influenza A virus-specificagents amantadine and rimantadine). Rapid detection of influenza virusis therefore essential to facilitate patient management and to initiateeffective control measures.

Presently known procedures for preparing a specimen for DirectFluorescence Antibody (DFA) staining are expensive, laborious and timeconsuming. Usually, a drop of a cell suspension from the specimen isdried on a glass slide and fixed with a precipitating or denaturingfixative such as acetone, methanol and ethanol. These compounds act toreduce the solubility of protein molecules and by disrupt proteintertiary hydrophobic interactions. After fixation, the samples arestained with fluorescent antibodies involving several steps: i)labelling; ii) washing; and iii) adhering a coverslip. Finally, thesamples are examined under a fluorescence microscope.

Further problems in DFA techniques are encountered during themicroscopic examination because the antibody preparations commonlycontain a general protein counter stain, such as Evans Blue, to help inidentifying cells. This counter stain also stains non-cellular materialwhich can make identifying cells difficult. Further, if the cells arenot completely dry, they can be lost during the processing steps,leading to an inadequate number of cells to make a judgment as to thepresence of the virus. Current DFA methods also require a highly skilledtechnician to prepare, read and interpret results because of thenon-specific staining mucus or debris that can be found in the specimen.Cell morphology and staining patterns are also compromised when thecells are dried onto the glass.

What is needed in the art is an improved DFA assay with better accuracyand faster processing time than those currently available.

SUMMARY

This invention is related to processing biological samples for directvirus detection in a liquid format. For example, the sample may bederived from the respiratory system (e.g., a lung aspirate ornasopharyngeal swab sample). The detection method may use antibodiesthat directly bind to a viral antigen on or in a cell, thereby allowingidentification as well as detection. In some instances, the antibodiesare labeled monoclonal antibodies. The method may be integrated with adevice comprising an algorithm capable of differentiating between aplurality of fluorescent signals.

In one embodiment, the present invention contemplates a method toperform a liquid direct fluorescent assay (LDFA) comprising at least onefluorescent label. In one embodiment, the fluorescent label comprisesR-phycoerythrin (PE). In one embodiment, the fluorescent label comprisesfluorescein isothiocyanate (FITC). In one embodiment, the fluorescentlabel is attached to an antibody.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a biological sample comprising at least oneviral antigen; ii) first and second antibodies, wherein said firstantibody reacts with a first viral antigen and does not react with asecond viral antigen and is labeled with a first fluorescent tag, andwherein said second antibody reacts with said second viral antigen anddoes not react with said first viral antigen and is labeled with asecond fluorescent tag; b) incubating at least a portion of said samplewith said first and second antibodies in a suspension under conditionssuch that only one of said first and second antibodies bind saidantigens; c) identifying a first virus based on detecting said firstfluorescent tag. In one embodiment, the method further comprises, step(d) identifying a second virus based on detecting said secondfluorescent tag. In one embodiment, the method further comprisesidentifying said first virus and said second virus based on detectingsaid first fluorescent tag and said second fluorescent tag. In oneembodiment, the first label comprises R-phycoerythrin. In oneembodiment, the second label comprises fluorescein isothiocyanate. Inone embodiment, the antibody comprises a monoclonal antibody. In oneembodiment, the incubating of the first and second antibodies with thesuspension is simultaneous. In one embodiment, the incubating of thefirst and second antibodies with the suspension is serial. In oneembodiment, the virus may be selected from the group including, but notlimited to, rhinovirus, human papilloma virus, human immunodeficiencyvirus, hepatitis virus, Newcastle disease virus, cardiovirus,corticoviridae, cystoviridae, epstein-barr virus, filoviridae,hepadnviridae, hepatitis virus, herpes virus, influenza virus,inoviridae, iridoviridae, metapneumovirus, orthomyxoviridae,papovavirus, paramyxoviridae, parvoviridae, polydnaviridae, poxyviridae,reoviridae, rhabdoviridae, semliki forest virus, tetraviridae,toroviridae, varicella zoster virus, vaccinia virus, and vesicularstomatitis virus.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a biological sample comprising cellsinfected with at least one viral antigen; ii) first and secondantibodies, wherein said first antibody reacts with a respiratorysyncytial viral antigen and does not react with a metapneumovirus viralantigen and is labeled with a first fluorescent tag, and wherein saidsecond antibody reacts with the metapneumovirus viral antigen and doesnot react with the respiratory syncytial viral antigen and is labeledwith a second fluorescent tag; b) incubating at least a portion of saidsample with said first and second antibodies in a suspension underconditions such that only one of said first and second antibodies bindssaid antigens; and c) identifying the viral antigen based on detectingthe first or second fluorescent tag. In one embodiment, the methodidentifies the respiratory viral antigen based on detecting the firstfluorescent tag. In one embodiment, the method identifies themetapneumovirus viral antigen based on detecting the second fluorescenttag. In one embodiment, the method identifies the respiratory syncytialviral antigen and the metapneumovirus viral antigen based on detectingthe first and second fluorescent tags. In one embodiment, the firstlabel comprises R-phycoerythrin. In one embodiment, the second labelcomprises fluorescein isothiocyanate. In one embodiment, the antibodycomprises a monoclonal antibody. In one embodiment, the incubating ofthe first and second antibodies with the suspension is simultaneous. Inone embodiment, the incubating of the first and second antibodies withthe suspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a biological sample comprising at least oneviral antigen; ii) first and second antibodies, wherein said firstantibody reacts with an influenza A viral antigen and does not reactwith an influenza B viral antigen and is labeled with a firstfluorescent tag, and wherein said second antibody reacts with saidinfluenza B viral antigen and does not react with said influenza A viralantigen and is labeled with a second fluorescent tag; b) incubating atleast a portion of said sample with said first and second antibodies ina suspension under conditions such that only one of said first andsecond antibodies binds said virus; and c) identifying the at least oneviral antigen based on detecting the first or second fluorescent tag. Inone embodiment, the method identifies the influenza A viral antigenbased on detecting the first fluorescent tag. In one embodiment, themethod identifies the influenza B viral antigen based on detecting thesecond fluorescent tag. In one embodiment, the method identifies theinfluenza A viral antigen and the influenza B viral antigen based ondetecting the first and second fluorescent tags. In one embodiment, thefirst label comprises R-phycoerythrin. In one embodiment, the secondlabel comprises fluorescein isothiocyanate. In one embodiment, theantibody comprises a monoclonal antibody. In one embodiment, theincubating of the first and second antibodies with the suspension issimultaneous. In one embodiment, the incubating of the first and secondantibodies with the suspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a biological sample comprising at least oneviral antigen; ii) first and second antibodies, wherein said firstantibody reacts with a parainfluenza viral antigen and does not reactwith an adenovirus viral antigen and is labeled with a first fluorescenttag, and wherein said second antibody reacts with said adenovirus viralantigen and does not react with said parainfluenza viral antigen and islabeled with a second fluorescent tag; b) incubating at least a portionof said sample with said first and second antibodies in a suspensionunder conditions such that only one of said first and second antibodiesbinds said virus; and c) identifying the at least one viral antigenbased on detecting the first or second fluorescent tag. In oneembodiment, the method identifies the parainfluenza viral antigen basedon detecting the first fluorescent tag. In one embodiment, the methodidentifies the adenovirus viral antigen based on detecting the secondfluorescent tag. In one embodiment, the method identifies theparainfluenza viral antigen and the adenovirus viral antigen based ondetecting the first and second fluorescent tags. In one embodiment, thefirst label comprises R-phycoerythrin. In one embodiment, the secondlabel comprises fluorescein isothiocyanate. In one embodiment, theantibody comprises a monoclonal antibody. In one embodiment, theincubating of the first and second antibodies with the suspension issimultaneous. In one embodiment, the incubating of the first and secondantibodies with the suspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein the sample is suspected of comprising at least one viralantigen; ii) at least two fluorescently labeled antibodies, wherein saidat least one antigen is capable of interacting with at least one of saidfluorescently labeled antibodies, wherein said antibodies aredifferentially labeled; b) incubating said suspension with saidfluorescently labeled antibodies under conditions such that at least oneof said fluorescently labeled antibodies binds said at least one viralantigen, thereby forming a labeled antigen-antibody complex; and c)detecting said labeled antigen-antibody complex within said suspensionby identifying one fluorescently labeled antibody, thereby identifyingthe at least one virus antigen. In one embodiment, the biological sampleis derived from a patient, thereby diagnosing a virus infection. In oneembodiment, the fluorescently labeled antibody comprises a monoclonalantibody. In one embodiment, the viral antigen comprises a respiratorysyncytial virus viral antigen. In one embodiment, the fluorescentlylabeled monoclonal antibody comprises specific affinity for therespiratory syncytial virus viral antigen. In one embodiment, thefluorescently labeled monoclonal respiratory virus antibody comprises aPE fluorescent label. In one embodiment, the viral antigen comprises aninfluenza virus viral antigen. In one embodiment, the influenza virusviral antigen comprises an influenza A virus viral antigen. In oneembodiment, the influenza virus viral antigen comprises an influenza Bvirus viral antigen. In one embodiment, the fluorescently labeledantibody comprises a monoclonal antibody. In one embodiment, thefluorescently labeled monoclonal antibody comprises specific affinityfor the influenza A virus viral antigen. In one embodiment, thefluorescently labeled influenza A monoclonal antibody comprises a PEfluorescent label. In one embodiment, the fluorescently labeledmonoclonal antibody comprises specific affinity for the influenza Bvirus viral antigen. In one embodiment, the fluorescently labeledinfluenza B monoclonal antibody comprises a FTIC fluorescent label. Inone embodiment, the viral antigen comprises an adenovirus viral antigen.In one embodiment, the fluorescently labeled monoclonal antibodycomprises specific affinity for the adenovirus viral antigen. In oneembodiment, the fluorescently labeled adenovirus monoclonal antibodycomprises a FITC fluorescent label. In one embodiment, the viral antigencomprises a parainfluenza virus viral antigen. In one embodiment, theparainfluenza virus viral antigen comprises a parainfluenza 1 virusviral antigen. In one embodiment, the parainfluenza virus viral antigencomprises a parainfluenza 2 virus viral antigen. In one embodiment, theparainfluenza virus viral antigen compries a parainfluenza 3 virus viralantigen. In one embodiment, the fluorescently labeled monoclonalantibody comprises specific affinity for the parainfluenza virus. In oneembodiment, the fluorescently labeled parainfluenza monoclonal antibodycomprises a PE fluorescent label. In one embodiment, the fluorescentlylabeled parainfluenza monoclonal antibody comprises specific affinityfor the parainfluenza 1 virus viral antigen. In one embodiment, thefluorescently labeled parainfluenza monoclonal antibody comprisesspecific affinity for the parainfluenza 2 virus viral antigen. In oneembodiment, the fluorescently labeled parainfluenza monoclonal antibodycomprises specific affinity for the parainfluenza 3 virus viral antigen.In one embodiment, the viral antigen comprises a metapneumovirus viralantigen. In one embodiment, the fluorescently labeled monoclonalantibody comprises a specific affinity for the metapnuemovirus viralantigen. In one embodiment, the fluorescently labeled metapneumovirusmonoclonal antibody comprises a FITC fluorescent label. In oneembodiment, the viral antigen comprises a varicella zoster viralantigen. In one embodiment, the fluorescently labeled monoclonalantibody comprises a specific affinity for the varicella zoster viralantigen. In one embodiment, the fluorescently labeled varicella zostermonoclonal antibody comprises a PE fluorescent label. In one embodiment,the viral antigen comprises a herpes simplex viral antigen. In oneembodiment, the fluorescently labeled monoclonal antibody comprises aspecific affinity for a herpes simplex-1 viral antigen. In oneembodiment, the fluorescently labeled monoclonal antibody comprises aspecific affinity for a herpes simplex-2 viral antigen. In oneembodiment, the fluorescently labled herpes simplex monoclonal antibodycomprises a FITC fluorescent label. In one embodiment, the suspensionincludes a staining reagent selected from the group of Evans blue,propidium iodide, acridine orange and combinations thereof. In oneembodiment, the suspension includes a detergent. In one embodiment, thedetergent is sapogenin. In one embodiment, the incubating of thefluorescently labeled antibodies and suspension is simultaneous. In oneembodiment, the incubating of the fluorescently labeled antibodies andsuspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein said sample is suspected of comprising a respiratorysyncytial virus viral antigen; ii) at least two fluorescently labeledantibodies, wherein said viral antigen is capable of interacting with atleast one of said fluorescently labeled antibodies, wherein antibodiesare differentially labeled; b) incubating said suspension with saidfluorescently labeled antibodies under conditions such that saidrespiratory syncytial virus viral antigen binds to at least one of saidfluorescently labeled antibodies, thereby forming a labeledantigen-antibody complex; and c) detecting said labeled antigen-antibodycomplex within said suspension by identifying one fluorescent labeledantibody, thereby identifying said respiratory syncytial virus viralantigen. In one embodiment, the biological sample is derived from apatient, thereby diagnosing a respiratory syncytial virus infection. Inone embodiment, the fluorescently labeled antibody comprises amonoclonal antibody. In one embodiment, the fluorescently labeledmonoclonal antibody comprises specific affinity for the respiratorysyncytial virus viral antigen. In one embodiment, the fluorescentlylabeled monoclonal respiratory virus antibody comprises a PE fluorescentlabel. In one embodiment, the suspension includes a staining reagentselected from the group of Evans blue, propidium iodide, acridine orangeand combinations thereof. In one embodiment, the suspension includes adetergent. In one embodiment, the detergent is sapogenin. In oneembodiment, the respiratory syncytial virus monoclonal antibody isderived from a clone selected from the group comprising clone 3A4D9 orclone 4F9G3. In one embodiment, the incubating of the fluorescentlylabeled antibodies and suspension is simultaneous. In one embodiment,the incubating of the fluorescently labeled antibodies and suspension isserial.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein said sample is suspected of comprising a influenza virusviral antigen; ii) at least two fluorescently labeled antibodies,wherein said viral antigen is capable of interacting with at least oneof said fluorescently labeled antibodies, wherein antibodies aredifferentially labeled; b) incubating said suspension with saidfluorescently labeled antibodies under conditions such that at least oneof said fluorescently labeled antibodies binds to the influenza virusviral antigen, thereby forming a labeled antigen-antibody complex; andc) detecting said labeled antigen-antibody complex within saidsuspension by identifying one fluorescently labeled antibody, therebyidentifying said influenza virus viral antigen. In one embodiment, thebiological sample is derived from a patient, thereby diagnosing aninfluenza virus infection. In one embodiment, the influenza virus viralantigen comprise an influenza A virus viral antigen. In one embodiment,the influenza virus viral antigen comprise an influenza B virus viralantigen. In one embodiment, the fluorescently labeled antibody comprisesa monoclonal antibody. In one embodiment, the fluorescently labeledmonoclonal antibody comprises specific affinity for the influenza Avirus viral antigen. In one embodiment, the fluorescently labeledinfluenza A monoclonal antibody comprises a PE fluorescent label. In oneembodiment, the fluorescently labeled monoclonal antibody comprisesspecific affinity for the influenza B virus viral antigen. In oneembodiment, the influenza B monoclonal antibody is derived from a cloneselected from the group comprising clone 8C7E11 or clone 9B4D9. In oneembodiment, the fluorescently labeled influenza B monoclonal antibodycomprises a FTIC fluorescent label. In one embodiment, the suspensionincludes a staining reagent selected from the group of Evans blue,propidium iodide, acridine orange and combinations thereof. In oneembodiment, the suspension includes a detergent. In one embodiment, thedetergent is sapogenin. In one embodiment, the influenza A monoclonalantibody is derived from a clone selected from the group comprisingclone 2H3C5 or clone A(6)B11. In one embodiment, the incubating of thefluorescently labeled antibodies and suspension is simultaneous. In oneembodiment, the incubating of the fluorescently labeled antibodies andsuspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein said sample is suspected of having an adenovirus viralantigen; ii) at least two fluorescently labeled antibodies, wherein saidviral antigen is capable of interacting with at least one of saidfluorescently labeled antibodies, wherein antibodies are differentiallylabeled; b) incubating said suspension with said fluorescently labeledantibodies under conditions such that said at least one of saidfluorescently labeled antibodies binds to said adenovirus viral antigen,thereby forming a labeled antigen-antibody complex; and c) detectingsaid labeled antigen-antibody complex within said suspension byidentifying one fluorescently labeled antibody, thereby identifying saidadenovirus viral antigen. In one embodiment, the biological sample isderived from a patient, thereby diagnosing an adenovirus infection. Inone embodiment, the fluorescently labeled antibody comprises amonoclonal antibody. In one embodiment, the fluorescently labeledmonoclonal antibody comprises specific affinity for the adenovirus viralantigen. In one embodiment, the fluorescently labeled adenovirusmonoclonal antibody comprises a FITC fluorescent label. In oneembodiment, the suspension includes a staining reagent selected from thegroup of Evans blue, propidium iodide, acridine orange and combinationsthereof. In one embodiment, the suspension includes a detergent. In oneembodiment, the detergent is sapogenin. In one embodiment, theadenovirus monoclonal antibody is derived from a clone selected from thegroup comprising clone 8H2C9, clone 2H10E2, or clone 4H6C9. In oneembodiment, the incubating of the fluorescently labeled antibodies andsuspension is simultaneous. In one embodiment, the incubating of thefluorescently labeled antibodies and suspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein the sample is suspected of comprising a parainfluenzavirus viral antigen; ii) at least two fluorescently labeled antibodies,wherein said viral antigen is capable of interacting with at least oneof said fluorescently labeled antibodies, wherein the antibodies aredifferentially labeled; b) incubating said suspension with saidfluorescently labeled antibody under conditions such that said at leastone of said fluorescently labeled antibodies binds to said parainfluenzavirus viral antigen, thereby forming a labeled antigen-antibody complex;and c) detecting said labeled antigen-antibody complex by identifyingone fluorescently labeled antibody, thereby identifying theparainfluenza virus viral antigen. In one embodiment, the biologicalsample is derived from a patient, thereby diagnosing a parainfluenzavirus infection. In one embodiment, the influenza virus viral antigencomprise a parainfluenza 1 virus viral antigen. In one embodiment, theinfluenza virus viral antigen comprise a parainfluenza 2 virus viralantigen. In one embodiment, the influenza virus viral antigen comprise aparainfluenza 3 virus viral antigen. In one embodiment, thefluorescently labeled antibody comprises a monoclonal antibody. In oneembodiment, the fluorescently labeled monoclonal antibody comprises a PEfluorescent label. In one embodiment, the fluorescently labeledmonoclonal antibody comprises specific affinity for a parainfluenza 1virus viral antigen. In one embodiment, the fluorescently labeledmonoclonal antibody comprises specific affinity for a parainfluenza 2virus viral antigen. In one embodiment, the fluorescently labeledmonoclonal antibody comprises specific affinity for a parainfluenza 3virus viral antigen. In one embodiment, the suspension includes astaining reagent selected from the group of Evans blue, propidiumiodide, acridine orange and combinations thereof. In one embodiment, thesuspension includes a detergent. In one embodiment, the detergent issapogenin. In one embodiment, the parainfluenza 1 monoclonal antibody isderived from a clone selected from the group comprising 1D8E10 or9F61C9. In one embodiment, the parainfluenza 2 monoclonal antibody isderived from a clone selected from the group comprising clone 2E4D7 orclone 5E4E11. In one embodiment, the parainfluenza 3 monoclonal antibodyis derived from a clone selected from the group comprising clone4G5(1)E2H9 or clone 1F6C8. In one embodiment, the incubating of thefluorescently labeled antibodies and suspension is simultaneous. In oneembodiment, the incubating of the fluorescently labeled antibodies andsuspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein the sample is suspected of comprising a metapnuemovirusviral antigen; ii) at least two fluorescently labeled antibodies,wherein said viral antigen is capable of interacting with saidfluorescently labeled antibodies, wherein antibodies are differentiallylabeled; b) incubating said suspension with said fluorescently labeledantibodies under conditions such that at least one of said fluorescentlylabeled antibodies binds to said metapneumovirus viral antigen, therebyforming a labeled antigen-antibody complex; and c) detecting saidlabeled antigen-antibody complex by identifying one fluorescentlylabeled antibody, thereby identifying the metapnuemovirus viral antigen.In one embodiment, the biological sample is derived from a patient,thereby diagnosing an metapneumovirus infection. In one embodiment, thefluorescently labeled antibody comprises a monoclonal antibody. In oneembodiment, the fluorescently labeled monoclonal antibody comprises aspecific affinity for the metapnueovirus viral antigen. In oneembodiment, the fluorescently labeled metapneumovirus monoclonalantibody comprises a FITC fluorescent label. In one embodiment, thesuspension includes a staining reagent selected from the group of Evansblue, propidium iodide, acridine orange and combinations thereof. In oneembodiment, the suspension includes a detergent. In one embodiment, thedetergent is sapogenin. In one embodiment, the metapneumovirusmonoclonal antibody is derived from a clone selected from the groupcomprising clone #4, clone #23, or clone #28. In one embodiment, theincubating of the fluorescently labeled antibodies and suspension issimultaneous. In one embodiment, the incubating of the fluorescentlylabeled antibodies and suspension is serial.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein the sample comprises unfixed cells derived from saidpatient, said suspension further comprising sapogenin and lackingfixatives and non-aqueous solvents; and ii) a fluorescently labeledantibody reactive with a viral antigen; and b) introducing saidfluorescently labeled antibody into said cell suspension underconditions such that at least a portion of said antibody reacts withsaid viral antigen, thereby revealing the viral antigen with said cells.In one embodiment, the sample is derived from a patient suspected ofhaving a virus infection. In one embodiment, the viral antigen isintracellular. In one embodiment, the viral antigen is extracellular. Inone embodiment, the viral antigen is attached to a virus. In oneembodiment, the viral antigen is displayed on the cell surface.

In one embodiment, the present invention contemplates a cytometer,comprising: a) a sample container configured to reside within a sampletray, wherein said tray is slidably engaged with said cytometer; b) anexcitation illumination source positioned to illuminate at least aportion of said container; and c) a detector positioned to collect anemission illumination from said at least a portion of said container. Inone embodiment, the sample container comprises a microscope slide havinga plurality of wells. In one embodiment, the sample tray slides toserially expose said plurality of containers to said illuminatedportion. In one embodiment, the excitation illumination source compriseslight emitting diodes. In one embodiment, the emission illumination isderived from a fluorescently labeled monoclonal antibody.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a suspension comprising a biologicalsample, wherein said sample comprises fluorescently labeled biologicalcells; ii) a cytometer comprising a sample tray, wherein said tray isconfigured to translate a sample container within said device, whereinsaid container comprises a plurality of samples; iii) an excitationillumination source targeted to said at least one sample; and b)inserting said sample container into said sample tray under conditionssuch that a first sample is illuminated by said excitation illuminationsource; and c) translating said sample container such that a secondsample is illuminated by said illumination source. In one embodiment,the fluorescently labeled cell comprises a fluorescent dye. In oneembodiment, the fluorescent dye is selected from the group consisting ofpropidium iodide, ethidium bromide and acridine orange. In oneembodiment, the fluorescently labeled cell comprises a fluorescentlylabeled monoclonal antibody. In one embodiment, the fluorescentlylabeled antibody comprises R-phycoerythrin. In one embodiment, thefluorescently labeled antibody comprises fluorescein isothiocyanate.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a suspension comprising a biologicalsample, wherein said sample comprises at least two fluorescently labeledviral antigens; and ii) a cytometer capable of differentially detectingthe fluorescently labeled viral antigens; b) placing said suspensioninto said cytometer; and c) detecting at least one of said fluorescentlylabeled viral antigens. In one embodiment, the detection of a firstviral antigen identifies a first virus. In one embodiment, the detectionof a second viral antigen identifies a second virus.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a suspension comprising a biologicalsample, wherein the sample is suspected of comprising diseased cells;ii) at least two fluorescently labeled antibodies, wherein said cellsare capable of interacting with at least one of said fluorescentlylabeled antibodies, wherein said antibodies are differentially labeled;and c) incubating said suspension with said fluorescently labeledantibodies under conditions such that at least one of said fluorescentlylabeled antibodies binds to said cells, thereby forming a labeledcell-antibody complex; and d) detecting said labeled cell-antibodycomplex within said suspension by identifying one fluorescently labeledantibody, thereby diagnosing said diseased cells. In one embodiment, thebiological sample is derived from a patient. In one embodiment, thesuspension includes a staining reagent selected from the group of Evansblue, propidium iodide, acridine orange and combinations thereof. In oneembodiment, the suspension includes a detergent. In one embodiment, thefluorescently labeled antibody comprises R-phycoerythrin (PE). In oneembodiment, the fluorescently labeled antibody comprises fluoresceinisothiocyanate (FITC). In one embodiment, the detergent is sapogenin. Inone embodiment, the incubating of the fluorescently labeled antibodiesand suspension is simultaneous. In one embodiment, the incubating of thefluorescently labeled antibodies and suspension is serial.

Definitions

The term “suspected of” as used herein, refers to a medical condition orset of medical conditions exhibited by a patient that suggest that thepatient may contract a particular disease or affliction. For example,these conditions may include, but are not limited to, unusual physicalsymptoms, unusual emotional symptoms, or unusual biochemical testresults.

The term “a liquid cell suspension” or “suspension” as used hereinrefers to any fluid composition comprising a biological sample, whereinthe components of the sample remain mobile relative to any natural orartificial surfaces and/or substrates. The fluid may comprise aqueouscomponents as well as organic components. For example, a liquid cellsuspension may comprise phosphate buffered saline.

The term “attached” as used herein, refers to any interaction between amedium (or carrier) and a drug. Attachment may be reversible orirreversible. Such attachment includes, but is not limited to, covalentbonding, ionic bonding, Van der Waals forces or friction, and the like.A drug is attached to a medium (or carrier) if it is impregnated,incorporated, coated, in suspension with, in solution with, mixed with,etc.

The term “derived from” as used herein, refers to the source of an itemof interest (i.e., for example, a monoclonal antibody or an energysignature). In one respect, a virus infected cell may be derived from abiological organism (i.e., for example, a human, animal, plant, orpatient). In one respect, a monoclonal antibody may be derived from ahybridoma clonal cell line (i.e., for example, a clone). In one respect,an emission illumination may be derived from a fluorescent compound. Inone respect, an excitation illumination may be derived from a lightsource.

The term “based on” as used herein, refers to any process or method,including a mathematical algorithm that results in the ability toquantitate the intensity of a specific excitation source. Further, theprocess, method, or mathematical algorithm is capable of differentiatingbetween a plurality of excitation sources such that they can beindividually quantitated and compared.

The term “detecting” or “detect” or “detected” as used herein, refers toany method and/or device that is capable of identifying an illuminationor excitation source.

The term “patient”, as used herein, is a human or animal and need not behospitalized. For example, out-patients, persons in nursing homes are“patients.” A patient may comprise any age of a human or non-humananimal and therefore includes both adult and juveniles (i.e., children).It is not intended that the term “patient” connote a need for medicaltreatment, therefore, a patient may voluntarily or involuntarily be partof experimentation whether clinical or in support of basic sciencestudies.

The term “affinity” as used herein, refers to any attractive forcebetween substances or particles that causes them to enter into andremain in chemical combination. For example, an inhibitor compound thathas a high affinity for a receptor will provide greater efficacy inpreventing the receptor from interacting with its natural ligands, thanan inhibitor with a low affinity.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatconsist of amino acid residues joined by peptide bonds, contain theelements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general,a protein comprises amino acids having an order of magnitude within thehundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

“Nucleic acid sequence” and “nucleotide sequence” as used herein referto an oligonucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.

The term “an isolated nucleic acid”, as used herein, refers to anynucleic acid molecule that has been removed from its natural state(e.g., removed from a cell and is, in a preferred embodiment, free ofother genomic nucleic acid).

The terms “amino acid sequence” and “polypeptide sequence” as usedherein, are interchangeable and to refer to a sequence of amino acids.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

The term “antibody” refers to immunoglobulin evoked in animals by animmunogen (antigen). It is desired that the antibody demonstratesspecificity to epitopes contained in the immunogen. The term “polyclonalantibody” refers to immunoglobulin produced from more than a singleclone of plasma cells; in contrast “monoclonal antibody” refers toimmunoglobulin produced from a single clone of plasma cells. Allmonoclonal antibodies contemplated herein having specific affinity for aviral antigen are commercially available. (Diagnostics Hybrids, Inc.,Athens, Ohio).

The terms “specific affinity”, “specific binding” or “specificallybinding” when used in reference to the interaction of an antibody and aprotein or peptide means that the interaction is dependent upon thepresence of a particular structure (i.e., for example, an antigenicdeterminant or epitope) on a protein; in other words an antibody isrecognizing and binding to a specific protein structure rather than toproteins in general. For example, if an antibody is specific for epitope“A”, the presence of a protein containing epitope A (or free, unlabelledA) in a reaction containing labeled “A” and the antibody will reduce theamount of labeled A bound to the antibody.

The term “sample” as used herein, is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., nasopharyngealdischarge, blood, plasma and serum), solid (e.g., stool), tissue, liquidfoods (e.g., milk), and solid foods (e.g., vegetables). For example, apulmonary sample may be collected by bronchoalveolar lavage (BAL) whichcomprises fluid and cells derived from lung tissues. A biological samplemay be collected that is suspected of containing a virus-infected cell,tissue extract, or body fluid.

The term “immunologically active” defines the capability of a natural,recombinant or synthetic peptide (i.e., for example, a collagen-likefamily protein), or any oligopeptide thereof, to induce a specificimmune response in appropriate animals or cells and/or to bind withspecific antibodies.

The term “antigenic determinant” as used herein, refers to that portionof a molecule that is recognized by a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody. One such antigenicdeterminant may be “a viral antigen” wherein an antigen may be displayedon, or within, a virus-infected host cell surface or on a virus coatsurface.

The terms “immunogen,” “antigen,” “immunogenic” and “antigenic” refer toany substance capable of generating antibodies when introduced into ananimal. By definition, an immunogen must contain at least one epitope(the specific biochemical unit capable of causing an immune response),and generally contains many more. Proteins are most frequently used asimmunogens, but lipid and nucleic acid moieties complexed with proteinsmay also act as immunogens. The latter complexes are often useful whensmaller molecules with few epitopes do not stimulate a satisfactoryimmune response by themselves.

The term “antibody” refers to immunoglobulin evoked in animals by animmunogen (antigen). It is desired that the antibody demonstratesspecificity to epitopes contained in the immunogen. The term “polyclonalantibody” refers to immunoglobulin produced from more than a singleclone of plasma cells; in contrast “monoclonal antibody” refers toimmunoglobulin produced from a single clone of plasma cells.

The term “label” or “detectable label” are used herein, to refer to anycomposition detectable by fluorescence, spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means. Forexample, such labels may include, but are not limited to,tetramethylrhodamine isothiocyanate (TRITC), Quantum Dots, CY3 and CY5.Other such labels include, but are not limited to, biotin for stainingwith labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads®),fluorescent dyes (e.g., fluorescein, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and calorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads. Patents teaching the useof such labels include, but are not limited to, U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241 (all herein incorporated by reference). The labelscontemplated in the present invention may be detected by many methods.For example, radiolabels may be detected using photographic film orscintillation counters, fluorescent markers may be detected using aphotodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting, thereaction product produced by the action of the enzyme on the substrate,and calorimetric labels are detected by simply visualizing the coloredlabel.

The term “binding” as used herein, refers to any interaction between aninfection control composition and a surface. Such as surface is definedas a “binding surface”. Binding may be reversible or irreversible. Suchbinding may be, but is not limited to, non-covalent binding, covalentbonding, ionic bonding, Van de Waal forces or friction, and the like. Aninfection control composition is bound to a surface if it isimpregnated, incorporated, coated, in suspension with, in solution with,mixed with, etc.

The term “fluorescent focus” refers to either one cell or a group ofclosely adjacent cells that fluoresce when fluorescently labeledantibodies. Some single virus infections produce multi-cell plaques andothers result only with infections of one or two cells per viable virus.A viral plaque consisting of many fluorescent staining cells is countedas “one” for viruses such as HSV, VZV, and RSV. Viruses such asinfluenza A, B, and adenovirus produce only one or a few fluorescentstaining cells per viable infectious virus.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery). Viruses are exemplifiedby, but not limited to, adenovirus, rhinovirus, human papilloma virus,human immunodeficiency virus, hepatitis virus, Newcastle disease virus,cardiovirus, corticoviridae, cystoviridae, epstein-barr virus,filoviridae, hepadnviridae, hepatitis virus, herpes virus, influenzavirus, inoviridae, iridoviridae, metapneumovirus, orthomyxoviridae,papovavirus, parainfluenza virus, paramyxoviridae, parvoviridae,polydnaviridae, poxyviridae, reoviridae, respiratory syncytial virus,rhabdoviridae, semliki forest virus, tetraviridae, toroviridae, vacciniavirus, and vesicular stomatitis virus. “Virus” also includes an animalvirus that is not a plus-strand RNA virus as exemplified by, but notlimited to, Arenaviridae, Baculoviridae, Birnaviridae, Bunyaviridae,Cardiovirus, Corticoviridae, Cystoviridae, Epstein-Barr virus,Filoviridae, Hepadnviridae, Hepatitis virus, Herpesviridae, Influenzavirus, Inoviridae, Iridoviridae, Metapneumovirus, Orthomyxoviridae,Papovaviru, Paramyxoviridae, Parvoviridae, Polydnaviridae, Poxyviridae,Reoviridae, Rhabdoviridae, Semliki Forest virus, Tetraviridae,Toroviridae, Vaccinia virus, Vesicular stomatitis virus.

The term “pathogen” as used herein, refers to any submicroscopic ormicroscopic organism comprising at least one antigen. For example, apathogen comprising an antigen can be detected and identified by afluorescently labeled monoclonal antibody having specific affinity tothe pathogen antigen. Representative examples, of pathogens include, butare not limited to, bacteria, fungi, yeast, viruses, or any microbe.

The term “respiratory virus” as used herein, refers to any virus capableof infecting pulmonary tissues (i.e., for example, lung tissue). Forexample, a respirator virus includes, but is not limited to, influenza,parainfluenza, adenovirus, rhinovirus, herpes simplex virus, respiratorysyncytial virus, hantavirus, or cytomegalovirus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary data of fluorescently labeled monoclonal antibody(MAb) incubation in non-influenza A virus infected cells (i.e, anegative control) Yellow stain: Background signal.

FIG. 2 shows exemplary data of fluorescently labeled MAb incubation in a1+ dilution aliquot (low titer) of influenza A virus-infected cells.Apple green stain: MAb-labeled cells. Yellow stain: Background signal.

FIG. 3 shows exemplary data of fluorescently labeled MAb incubation in a4+ dilution aliquot (high titer) of influenza A virus-infected cells.Apple green stain: FITC MAb-labeled virus infected cells. Yellow stain:Background signal.

FIG. 4 shows exemplary data of fluorescently labeled FITC MAb incubationin adenovirus-infected cells. Upper Panel: Negative control specimenstained with propidium iodide and Evans blue. Lower Panel: Apple greenstain: FITC MAb-labeled adenoirus infected cell.

FIG. 5 shows exemplary data of fluorescently labeled PE MAb incubationin respiratory virus-infected cells. Upper Panel: Negative controlspecimen stained with propidium iodide and Evans blue. Lower Panel: Goldstain: PE MAb-labeled adenoirus infected cell.

FIG. 6 shows exemplary data of an SDS-PAGE electropherogram isolation ofinfluenza A virus MAbs. 0 MAb A(6)B11: lanes A2, C5, and C6; ii) MAb10B12C11: lane B1; and iii) MAb 2H3C5: lanes C1, C2, and C3. Molecularweight markers are in lanes A6, B4, and C4; the 2 heavier marker bandsrepresent 50 and 20 kDa. respectively. Other lanes are representative ofother viral MAbs. Approximately 5-μg of protein were loaded onto eachwell.

FIG. 7 presents exemplary data of binding affinities of variousembodiments for Influenza A virus MAbs to Influenza A (Texas) virus. Redsquares: MAb 10B12C11. Blue circles: MAb 2H3C5. Green triangles: MAbZymeTx A(6)B11.

FIGS. 8A-B present exemplary data showing detection of influenza A virusinfected cells with a yellow-golden fluorescent monoclonal antibody(FIG. 8A) or an apple-green fluorescent monoclonal antibody (FIG. 8B).

FIGS. 9A-B present exemplary data showing a comparison of LDFA (MAbs:10B12C11+A(6)B11) versus DFA viral detection for influenza A (FIG. 9A)and influenza B (FIG. 9B).

FIGS. 10A-B present exemplary data showing a comparison of LDFA (MAbs:10B12C11+A(6)B11) versus DFA viral detection for respiratory virus (FIG.10A) and metapneumovirus (FIG. 10B).

FIGS. 11A-B present exemplary data showing a comparison of LDFA (MAbs:10B12C11+A(6)B11) versus DFA viral detection for adenovirus (FIG. 11A)and parainfluenza virus 1 (FIG. 11B).

FIGS. 12A-B present exemplary data showing a comparison of LDFA (MAbs:10B12C11+A(6)B11) versus DFA viral detection for parainfluenza virus 2(FIG. 12A) and parainfluenza virus 3 (FIG. 12B).

FIGS. 13A-B present exemplary data showing a comparison of LDFA (MAbs:10B12C11+A(6)B11) versus DFA viral detection for parainfluenza (1-3)(FIG. 13A); and a combined mixture of viruses in FIGS. 9-13A (FIG. 13B).

FIG. 14 presents one embodiment of a portable fluorescent reader capableof detecting and measuring emission illuminations from at least twodifferentially labeled MAbs. Also shown is a multi-well sample slidepositioned for entry into the device on a slide tray that is inserted(as a unit) into a sample drawer.

FIG. 15 presents one embodiment of a multi-well sample slide as shownwith the device of FIG. 14. Pipet indicates location of entry and exitports for the introduction and/or withdrawl of a liquid sample.

DETAILED DESCRIPTION OF THE INVENTION

This invention is related to processing biological samples for directvirus detection in a liquid format. For example, the sample may bederived from the respiratory system. The detection method may useantibodies that directly bind to a viral antigen, thereby allowingidentification as well as detection. In some instances, the antibodiesare labeled monoclonal antibodies. The method may be integrated with adevice comprising an algorithm capable of differentiating between aplurality of fluorescent signals.

In one embodiment, the present invention contemplates a method fordetecting and identifying a viral antigen using an image of a processedbiological cell specimen and an algorithm to determine if cells arepositive or negative for viral infection. In one embodiment, the methodcomprises a liquid sample during preparation, processing, andexamination.

I. Virus Infections

During epidemics, viruses may be a significant cause of morbidity andmortality, especially in the elderly and in patients with chronicpulmonary and/or cardiovascular disorders Swenson et al., “Rapiddetection of influenza virus in cell culture by indirectimmunoperoxidase staining with type-specific monoclonal antibodies”Diagn. Microbiol. Infect. Dis. 7:265-268 (1987). Appropriate infectioncontrol measures and proper patient management may be optimized by rapiddetection and identification of virus in clinical specimens.

A virus is a small infectious organism—much smaller than a fungus orbacterium—that must invade a living cell to reproduce (e.g., replicate).The virus attaches to a cell (called the host cell), enters it, andreleases its DNA or RNA inside the cell. The virus's DNA or RNA is thegenetic material containing the information needed to replicate thevirus. The virus's genetic material takes control of the cell and forcesit to replicate the virus. The infected cell usually dies because thevirus keeps it from performing its normal functions. When it dies, thecell releases new viruses, which go on to infect other cells.

Some viruses do not kill the cells they infect but instead alter thecell's functions. Sometimes the infected cell loses control over normalcell division and becomes cancerous. Some viruses leave their geneticmaterial in the host cell, where the material remains dormant for anextended time (e.g., latent infection). When the cell is disturbed, thevirus may begin replicating again and cause disease.

Viruses usually infect one particular type of cell. For example, coldviruses infect only cells of the upper respiratory tract. Additionally,most viruses infect only a few species of plants or animals. Some infectonly people. Many viruses commonly infect infants and children.

Viruses are spread (e.g., transmitted) in various ways. Some areswallowed, some are inhaled, and some are spread by the bites of insectsand other parasites (i.e., for example, mosquitoes and ticks). Some arespread sexually.

1. Defenses

Most biological organisms have a number of defenses against viruses. Forexample, physical barriers, such as the skin, discourage easy entry.Infected cells also make interferons, substances that can makeuninfected cells more resistant to infection by many viruses.

When a virus enters the body, the virus may trigger the body's immunedefenses. These defenses begin with white blood cells, such aslymphocytes and monocytes, which produce antibodies that attack anddestroy the virus or the infected cells. Production of antiviralantibodies produces a subsequent state of immunity, wherein the whiteblood cells are now programmed to immediately respond to re-infection.These states of immunity can be artificially induced by vaccination withnon-infectious viral particles. Vaccination initiates the production ofantibodies from a variety of white blood cells, thereby producingantibodies that are polyclonal in nature.

2. Types of Viral Infections

Probably the most common viral infections are those of the upperrespiratory airway (i.e., for example, nose, throat, etc.). Theseinfections include sore throat, sinusitis, and the common cold.Influenza is a viral respiratory infection. In small children, virusesalso commonly cause croup and inflammation of the windpipe (i.e., forexample, laryngotracheobronchitis) or other airways deeper inside thelungs. Respiratory infections are more likely to cause severe symptomsin infants, older people, and people with a lung or heart disorder.

Some viruses (i.e., for example, rabies virus, West Nile virus, andseveral different encephalitis viruses) infect the nervous system. Viralinfections also develop in the skin, sometimes resulting in warts orother blemishes.

Other common viral infections are caused by herpes viruses. Eightdifferent herpes viruses infect people, including but not limited to,herpes simplex virus type 1, herpes simplex virus type 2, andvaricella-zoster virus cause infections that produce blisters on theskin or mucus membranes. Another herpes virus, Epstein-Barr virus,causes infectious mononucleosis. Cytomegalovirus is a cause of seriousinfections in newborns and in people with a weakened immune system.Cytomegalovirus can also produce symptoms similar to infectiousmononucleosis in people with a healthy immune system. Human herpesviruses 6 and 7 cause a childhood infection called roseola infantum.Human herpes virus 8 has been implicated as a cause of cancer (Kaposi'ssarcoma) in people with AIDS.

All of the herpes viruses cause lifelong infection because the virusremains within its host cell in a dormant (latent) state. Sometimes thevirus reactivates and produces further episodes of disease. Reactivationmay occur rapidly or many years after the initial infection.

3. Diagnosis

Common viral infections are usually diagnosed based on symptoms. Forinfections that occur in epidemics (i.e., for example, influenza), thepresence of other similar cases may help doctors identify a particularinfection. For other infections, blood tests and cultures (growingmicroorganisms in the laboratory from samples of blood, body fluid, orother material taken from an infected area) may be done. Blood may betested for antibodies to viruses or for antigens (proteins on or inviruses that trigger the body's defenses). Polymerase chain reaction(PCR) techniques may be used to make many copies of the viral geneticmaterial, enabling doctors to rapidly and accurately identify the virus.Tests are sometimes done quickly—for instance, when the infection is aserious threat to public health or when symptoms are severe. A sample ofblood or other tissues is sometimes examined with an electronmicroscope, which provides high magnification with clear resolution.

4. Treatment

Drugs that combat viral infections are called antiviral drugs. Manyantiviral drugs work by interfering with replication of viruses, such asdrugs used to treat human immunodeficiency virus (HIV) infection.Because viruses replicate inside cells using the cells' own metabolicfunctions, there are only a limited number of metabolic functions thatantiviral drugs can target. Therefore, antiviral drugs are difficult todevelop. Further, effective antiviral drugs can be toxic to human cells.Viruses can also develop resistance to antiviral drugs.

Other antiviral drugs strengthen the biological immune response to theviral infection. These drugs include several types of interferons,immunoglobulins, and vaccines. Interferon drugs are replicas ofnaturally occurring substances that slow or stop viral replication.Immune globulin is a sterilized solution of antibodies (also calledimmunoglobulins) collected from a group of people. Vaccines arematerials that help prevent infection by stimulating the body's naturaldefense mechanisms. Many immune globulins and vaccines are given beforeexposure to a virus to prevent infection. Some immune globulins and somevaccines, such as those for rabies and hepatitis B, are also used afterexposure to the virus to help prevent infection from developing orreduce the severity of infection. Immune globulins may also help treatsome established infections and also prevent infection after futureexposures to the virus.

Most antiviral drugs can be given by mouth. Some can also be given byinjection into a vein (intravenously) or muscle (intramuscularly). Someare applied as ointments, creams, or eye drops or are inhaled as apowder.

Antibiotics are not effective against viral infections, but if a personhas a bacterial infection in addition to a viral infection, anantibiotic is often necessary.

II. Viral Detection Assays

Infectious disease rates and immunization strategies continue to evolvein the United States and worldwide in response to societal needs,national defense, and evolutionary changes in the organisms producingdisease. Immunizations are performed to prevent many infections, whileprophylactic population screening is utilized for infections lackingeffective vaccines and for those diseases having a low enough incidencethat mass immunization is not deemed most efficacious.

The current method for diagnosis of disease, determining exposure tobiological materials such as pathogens, or monitoring immunizationstatus varies depending on the specific assay. Some methods employ an invivo assay. Others require a biological sample, such as blood or serum,to be obtained and tested. Tests performed usually are one of thenon-homogeneous type diagnostic methods such as enzyme-linkedimmunosorbant assay (hereinafter “ELISA”), radioimmunoassay (hereinafter“RIA”), or agglutination. All are surface-binding, heterogeneous assaysand require the antigen of interest to interact with a surface toachieve success, often at the expense of high non-specific binding andloss of specificity.

The embodiments described herein improve upon previously reportedimmunoassays by providing a totally liquid environment encompassing allsteps of the method.

A. Non-Fluorescent Antibody Assays

A general method believed capable of detecting viruses in solution wasreported using composite organic-inorganic nanoclusters displayingantibodies that capture fluorescently labeled infected cells. Sun etal., “Multiplexed Detection of Analytes in Fluid Solution,” UnitedStates Patent Publication No. 2007/0279626. Thenanocluster-antibody-cell complex is then subjected to FACS inconjunction with Raman analysis to determine the number of capturedinfected cells. A liquid-phase immunodiagnostic assay has been reportedthat generates a biochemical reporter when antigen/antibody complex isacted upon by a first and second enzyme. Clemmons et al., “Liquid-PhaseImmunodiagnostic Assay,” U.S. Pat. No. 5,637,473. Suggestedantigen/antibody complexes include various virus-related epitopes.Analyte detection from clinical samples of patients suspected of havinga disease was reported by reacting a sample with a nucleic acid-labeledbinding construct. The binding construct may be an antibody havingaffinity to an analyte. Once bound, the antibody/analyte complex isisolated and the nucleic acid label is amplified and identified toquantitate the captured analytes. Lawton, “Soluble Analyte Detection andAmplification,” U.S. Pat. No. 7,341,837; and United States PatentPublication No. 2005/0048500.

B. Indirect Immunofluorescence

Indirect immunofluorescence represents a method in which a firstunlabeled IgG antibody directed against a specific antigen is thendetected by use of a labeled (i.e., for example, fluorescently labeled)anti-IgG of the same species as the first antibody. For example, labeledgoat anti-rabbit IgG antibody can be used against a specific firstantibody that was raised in rabbits.

Flow cytometry by using FACS methodology has been used for monitoringintracellular influenza A replication by using fluorescently labeledmonoclonal antibodies directed to matrix protein I and nucleoprotein. Inthis system, adherent MDCK cells were first inoculated with viruscontaining sample, then fixed and dehydrated with ethanol andparaformaldehyde/ethanol. Schulze-Horsel et al., “Flow CytometricMonitoring of Influenza A Virus Infection in MDCK Cells During VaccineProduction,” BMC Biotechnol. 8:45 (2008); and Lonsdale et al., “A RapidMethod for Immunotitration of Influenza Viruses Using Flow Cytometry,”J. Virol. Methods, 110(1):67-71, (2003)).

In vivo antibody production was studied in mice infected with influenzavirus using a FACS immunofluorescence method. The data demonstrated thatB cells isolated from infected spleen cells did not undergo isotypeswitching from natural IgM isotypes to influenza-specific isotypesduring the course of the infection. Baumgarth et al., “Innate andAcquired Humoral Immunities to Influenza Virus are Mediated by DistinctArms of the Immune System,” PNAS 96:2250-2255 (1999).

Detection of influenza virus was compared between various processingmethods using cell culture-based indirect immunofluorescence stainingChamber slides, shell vials, standard virus isolation, and nasal washspecimens were all tested using monoclonal antibodies specific forantigens of either influenza A virus (i.e., matrix protein ornucleoprotein) or influenza B virus (i.e., nucleoprotein orhemagglutinin) Walls et al., “Characterization and evaluation ofmonoclonal antibodies developed for typing influenza A and influenza Bviruses” J. Clin. Microbiol. 23:240-245 (1986). These comparisonsindicated that indirect immunofluoresence tests were difficult tointerpret due to an abundance of mucus debris despite vigorous washingand, occasionally, inadequate numbers of intact cells. Stokes et al.,“Rapid Diagnosis of Influenza A and B by 24-h Fluorescent Focus Assays,”J. Clin. Microbiol. 26(7):1263-1266 (1988). Influenza infections mayalso be detected by capturing naturally produced antibodies within aclinical sample onto a surface coated with recombinantly producedinfluenza A M2 protein. Kendal et al., “Improved Expression of InfluenzaA M2 Protein in Baculovirus and Uses of M2 Protein,” WO/1993/003173.Influenza virus infection may also be detected using a sandwichimmunofluorescent assay where anti-influenza antiserum recognizing NP,M1, HA and NA protein were reacted with fixed and permeabilized HeLacells. The resultant protein-antibody complexes were visualized withFITC-labeled anti-rabbit IgG antibody. Shiratsuchi et al.,“Phosphatidylserine-Mediated Phagocytosis of Influenza A Virus-InfectedCells by Mouse Peritoneal Macrophages,” J. Virol. 74(19):9240-9244(2000).

Influenza virus was detected on tissue impression smears using unlabeledinfluenza A group-specific monoclonal antibody detected by an anti-mouseFITC secondary antibody. The method does not teach use of sapogenin, orpropidium iodide. Selleck et al., “Rapid Diagnosis of Highly PathogenicAvian Influenza Using Pancreatic Impression Smears,” Avian Diseases47(s3):1190-1195 (2002).

C. Direct Fluorescent Assays (DFAs)

Direct immunofluorescence comprises the use of a labeled reactant (i.e.,for example, an antibody) which both detects and indicates the presenceof an unlabeled reactant (i.e., for example, an antigen, viral epitope,or cell epitope). In some cases, the label comprises a fluorescentmolecule. In some cases, it is advantageous to use primary antibodiesdirectly labeled with a fluorescent molecule. This direct labelingdecreases the number of steps in the staining procedure and, moreimportantly, often avoids cross-reactivity and high background problems.

1. Non-Liquid Based DFA

Direct detection of viruses has been accomplished by using animmunofluorescence or enzyme-linked immunosorbent assay (ELISA).Direct-smear examinations by immunofluorescence are problematic due tolow sensitivity and non-specific background staining. Alternatively, ashell vial centrifugation assay has been adapted for detection of theinfluenza viruses. Espy et al., “Rapid detection of influenza virus byshell vial assay with monoclonal antibodies” J. Clin. Microbiol.24:677-679 (1986); and Stokes et al., “Rapid diagnosis of influenza Aand B by 24-h fluorescent focus assay” J. Clin. Microbiol. 26:1263-1266(1988).

Some cell culture based techniques to detect influenza A and influenza Bviruses in clinical respiratory specimens use Madin-Darby canine kidneycells, which are very sensitive to infection with influenza virus. Suchmethods take at least a week of incubation to observe the development ofcytopathic effects resulting from viral infection of the cell culture bythe sample. Frank et al., “Comparison of different tissue cultures forisolation and quantitation of influenza and parainfluenza viruses” J.Clin. Microbiol. 10:32-36 (1979); and Meguro et al., “Canine kidney cellline for isolation of respiratory viruses” J. Clin. Microbiol. 9:175-179(1979). Clinical specimen smears were also examined by using a directimmunofluorescence assay. These smears were subjected to several stepsto prepare and dry the sample on a microscope slide before viewing on amicroscope. Influenza was detected using FTIC-labeled antibodies alongwith counter staining with Evan's blue. This method is not enhanced byusing sapogenin to improve the detectable signal or using a combinationcounterstain with propidium iodide. Mills et al., “Detection ofInfluenza Virus by Centrifugal Inoculation of MDCK Cells and Stainingwith Monoclonal Antibodies,” J. Clin. Microbiol. 27(11):2505-2508(1989).

Currently, there are two (2) general methods (i.e., standard DFA andcytospin DFA) used for staining respiratory specimens directly usingfluorescent labeled antibodies to detect the presence of respiratoryviruses such as influenza A and B, respiratory syncytial virus, etc.These assay protocols are compared to one embodiment contemplated herein(i.e., for example, liquid DFA; LDFA) that is much faster. See, Table 1.

TABLE 1 Estimated time to results for one specimen using a DFA StandardDFA Cytospin DFA* Drying 30-60 minutes 5-10 minutes Fixing 10 minutes 10minutes Incubation 15-30 minutes 15-30 minutes Manipulation time 2minutes 2 minutes Total time to result 47-102 minutes 32-52 minutes*Cytospin is done only for the Screen. If the Cytospin preparation ispositive, the lab still has to run the standard 8 well ID slide whichtakes 47-102 minutes.

The current standard and cytospin DFAs require numerous and lengthylaboratory steps including, i) centrifugation to collect and concentratethe cells from the specimen (this step varies depending on thelaboratory. It could range from 10 minutes to up to 30 minutes ifmultiple rinses are performed); ii) drying the deposited cells on theslide; iii) fixing the cells using a dehydration agent (i.e., forexample, Acetone); iv) incubating the adhered, fixed cells withrespective fluorescein isothiocyanate (FITC) labeled Ab's at 37° C.; andv) manipulating the labeled/fixed cells for microscope viewing andexamination for the presence of fluorescent cells. One significantdrawback of the current DFAs is that the microscope viewing andexamination for fluorescently labeled cells is done manually (i.e., byvisual inspection). Further, as a single fluorescent label is usuallyused for each antibody, a separate sample must be processed in series inorder to detect the presence of each suspected virus.

Fixatives in the DFAs is usually a dehydration agent (i.e., for example,acetone) which immobilizes proteins, adheres cells to a glass slide andpermeabilizes the cells for entry of MAb's to react with intracellularantigen. Staining agents in the DFAs are usually directly labeled FITCMAb's for the viral antigens in combination with a protein stain (i.e.,for example, Evans Blue) for counter-staining the cells.

2. Liquid DFA (LDFA)

Currently available DFAs would require a different aliquot to detect andidentify each virus (i.e., eight aliquots total) using the lengthy andlaborious techniques described above. For example, non-liquid DFAsdetection of eight (8) viruses require thirty-seven (37) laboratorymanipulations. In contrast, an LDFA embodiment contemplated by thepresent invention comprises only fourteen (14) laboratory manipulationsusing the serial analysis of three aliquots of a liquid sample. In oneembodiment, the method further comprises a fourth aliquot of the liquidsample without any labeled monoclonal antibodies as a control.

Fluorescently labeled ligands (i.e., for example, small molecules,peptides) have been used in solution-based diagnostic assays bydetecting antibodies by measuring changes in fluorescence polarization.A fluorescently labeled ligand will undergo an alteration in molecularspin rate, thereby changing its emission pattern when the ligand bindswith a binding partner (i.e., for example, a labeled antigen bindingwith an antibody). For instance, the method may detect naturallyproduced antibodies in biological samples from patient that are infectedwith a microorganism (i.e., for example, bacteria or virus). Cullum etal., “Fluorescence Polarization Instruments and Methods For Detection ofExposure to Biological Materials By Fluorescence PolarizationImmunoassay of Saliva, Oral or Bodily Fluids,” U.S. Pat. No. 7,408,640(2008); and United States Patent Publication No. 2005/0095601 (bothherein incorporated by reference).

Solutions of fluorescently labeled monoclonal antibodies have beenstabilized with azo-compounds for use to identify Mycoplasma pneumoniain an ELISA format. The infected cells were immobilized to a microwellplate before incubation with the antibodies. These methods do not dependupon improved cell permeability (i.e., for example, by addition ofsapogenin) or counterstaining with propidium iodide, and does notcontemplate detection of viruses (i.e., for example, influenza).Sawayanagi et al., “Stable Antibody Solution and Method For Preparingthe Same,” U.S. Pat. No. 5,602,234 (1997)(herein incorporated byreference).

In one embodiment, the present invention contemplates a method toperform LDFA comprising incubating a liquid sample with apermeabilization agent and at least one cell stain. Although it is notnecessary to understand the mechanism of an invention, it is believedthat this is a distinct advantage over currently available non-liquidDFA's which perform the analogous steps of fixation and staining in twoseparate steps. In one embodiment, the permeabilization agent comprisesacetone. In one embodiment, the cell stain comprises a specific proteinstain (i.e., for example, Evans Blue) at approximately one-eigth theamount in currently available DFAs and a non-specific cell nuclei stain(i.e., for example, propidium iodide).

In one embodiment, the present invention contemplates a method toperform LDFA comprising preparing a liquid sample for examination inless than ten (10) minutes. In one embodiment, the method comprisesincubating the liquid sample at room temperature with a permeabilizationagent (i.e., for example, acetone) and at least one cell stain forapproximately five (5) minutes. In one embodiment, the method comprisesrinsing and centrifuging the permeabilized and stained liquid sample atroom temperature for approximately two (2) minutes. The LDFA hassignificant advantages over currently known DFA assays by significantlyimproving the ability of a laboratory technician to quickly identify andenumerate virus-infected cells in a liquid specimen. See, Table 2.

TABLE 2 Estimated time to results for one specimen using LDFA. LiquidDFA Drying none Fixing none Incubation 5 minutes Wash 2 minutesManipulation time 2 minutes Total time to result 9 minutes

No fixatives are necessary in LDFAs to adhere cells to a glass slide,but dehydration agents may be useful as a cell permeabilzation agent.Further, a detergent (i.e., for example, sapogenin) may be used tooptimally permeabilize the cells for entry of the MAb's to react withintracellular antigen. Staining agents in LDFAs are usually directlylabeled fluorescent MAb's for a viral antigen in combination with a lowconcentration of Evans Blue (i.e., for example, to quench fluorescentbackground staining) and propidium iodide, a fluorescent nuclear stain,used to help identify what a cell is in relation to the fluorescencefrom FITC and/or PE with the nuclear stains in cells.

Such labeling has been observed to be proportional to the number ofinfected cells (i.e., for example, infected with influenza A) present inthe test solution. See, FIGS. 1, 2, and 3 performed in accordance withExample I. Similar data was obtained with HSV-1 infected cells (data notshown). One advantage of the currently disclosed LDFA is that the cellsuspensions do not require drying or covering with a mounting fluid tofaciliate microscopic examination. Although a wash step is also notrequired, it is believed that an embodiment of the present inventionthat comprises a wash step will have a lower background signal. Thesepreliminary studies demonstrated very good sensitivity based on acomparison of the number of MAb-positive cells in the scraped suspensionto the stained monolayer.

The present LDFA method was compared to conventional DFA methodsdemonstrating the specificity and selectivity of the LDFA versus atraditional DFA for: i) Influenza A (Flu A) MAb combination of clone2H3C5 and clone A(6)B11; ii) influenza B (Flu B) MAb combination ofclone 8C7E11 and clone 9B4D9; iii) respiratory virus (RSV) MAbcombination of clone 3A4D9 and clone 4F9G; iv) metapneumovirus (MPV) MAbcombination of clone #4, clone #23, and clone #28; v) adenovirus (ADV)MAb combination of clone 8H2C9, clone 2H10E2, and clone 4H6C9; vi)parainfluenza (PIV) virus 1 MAb combination of clone 1D8E10 and clone9F61C9; vii) parainfluenza virus 2 MAb combination of clone 2E4D7 andclone 5E4E11; viii) parainfluenza virus 3 MAb combination of clone4G5(1)E2H9 and clone 1F6C8; ix) pooled parainfluenza 1-3 MAbs asdescribed above and x) combined mixture of i)-ix). Representativemicrographs show MAb-positive signals for LDFA vents DFA results. See,FIG. 4 and FIG. 5, respectively. Further, in a single MAb assay system,LDFA and DFA identification of virus-positive cells versusvirus-negative cells are compared. See, Tables 3 through 8 respectively.

TABLE 3 Cross-correlation between LDFA and DFA for Influenza A virusdetection and identification using the LDFA Influenza A&B reagentcompared to the Individual Influenza A reagent.. TABLE 3: Study Site 4 -D³ Ultra Duet R-PE identification of Influenza A virus positivespecimens D³ Ultra Final Identification Direct Specimen (Influenza Avirus) (637 Specimens) Pos Neg D³ Ultra Duet Flu A/Flu B Pos 46  2 Neg 1 588 Positive Percent Agreement 97.6% (PPA) (46/47) 95% CI- PPA 88.9,99.6% Negative Percent Agreement 99.7% (NPA) (588/590) 95% CI- NPA 98.8,99.9%

TABLE 4 Cross-correlation between LDFA and DFA for Influenza B virusdetection and identification using the LDFA Influenza A&B reagentcompared to the Individual Influenza B reagent. TABLE 4: Study Site 4 -D³ Ultra Duet FITC identification of Influenza B virus positivespecimens D³ Ultra Final Identification Direct Specimen (Influenza Bvirus) (637 Specimens) Pos Neg D³ Ultra Duet Flu A/Flu B Pos 197  4 Neg 1 435 Positive Percent Agreement 99.5% (PPA) (197/198) 95% CI- PPA97.2, 99.9% Negative Percent Agreement 99.1% (NPA) (435/439) 95% CI- NPA97.7, 99.7%

TABLE 5 Cross-correlation between LDFA and DFA for RSV detection andidentification using the LDFA Influenza RSV&MPV reagent compared to theIndividual RSV reagent. TABLE 5: Study Site 4 - D³ Ultra Duet R-PEidentification of RSV positive specimens D³ Ultra Final IdentificationDirect Specimen (RSV) (637 Specimens) Pos Neg D³ Ultra Duet RSV/MPV Pos29  0 Neg  0 608 Positive Percent Agreement 100% (PPA) (29/29) 95% CI-PPA 88.3, 100% Negative Percent Agreement 100% (NPA) (608/608) 95% CI-NPA 99.4, 100%

TABLE 6 Cross-correlation between LDFA and DFA for MPV detection andidentification using the LDFA Influenza RSV&MPV reagent compared to theIndividual MPV reagent. TABLE 6: Study Site 4 - D³ Ultra Duet FITCidentification of MPV positive specimens Direct Specimen D³ MPV DFAReagent (637 Specimens) Pos Neg D³ Ultra Duet RSV/MPV Pos 15 0 Neg  0622  Positive Percent Agreement (PPA) 100% (15/15) 95% CI-PPA 79.6, 100%Negative Percent Agreement (NPA) 100% (622/622) 95% CI-NPA 99.4, 100%

TABLE 7 Cross-correlation between LDFA and DFA for Parainfluenza virusdetection and identification using the LDFA Parainfluenzapool&Adenovirus reagent compared to the Individual Parainfluenzareagents. TABLE 7: Study Site 4 - D³ Ultra Duet R-PE identification ofParainfluenza virus 1, 2, and 3 positive specimens D³ Ultra FinalIdentification Direct Specimen (Parainfluenza) (637 Specimens) Pos NegD³ Ultra Duet PIV/Adeno Pos 6 0 Neg 0 631  Positive Percent Agreement(PPA) 100% (6/6) 95% CI-PPA 56.6, 100% Negative Percent Agreement (NPA)100% (631/631) 95% CI-NPA 99.4, 100%

TABLE 8 Cross-correlation between LDFA and DFA for Adenovirus detectionand identification using the LDFA Influenza Parainfluenzapool&Adenovirus reagent compared to the Individual Adenovirus reagent..TABLE 8: Study Site 4 - D³ Ultra Duet FITC identification of Adenoviruspositive specimens D³ Ultra Final Identification Direct Specimen(Adenovirus) (637 Specimens) Pos Neg D³ Ultra Duet PIV/Adeno Pos 1 0 Neg0 636  Positive Percent Agreement (PPA) % (1/1) 95% CI-PPA 20.7, 100%Negative Percent Agreement (NPA) 100% (636/636) 95% CI-NPA 99.4, 100%

Studies have also demonstrated the specificity and selectivity of theLDFA versus a traditional DFA for: i) Influenza A (Flu A) MAbcombination of clone 10B12C11 and clone A(6)B11 (FIG. 9A); ii) influenzaB (Flu B) MAb combination of clone 8C7E11 and clone 9B4D9 (FIG. 9B);iii) respiratory syncytial virus (RSV) MAb combination of clone 3A4D9and clone 4F9G3 (FIG. 10A); iv) metapneumovirus (MPV) MAb combination ofclone #4, clone #23, and clone #28 (FIG. 10B); v) adenovirus (ADV) MAbcombination of clone 8H2C9, clone 2H10E2, and clone 4H6C9 (FIG. 11A);vi) parainfluenza (PIV) virus 1 MAb combination of clone 1D8E10 andclone 9F61C9 (FIG. 11B); vii) parainfluenza virus 2 MAb combination ofclone 2E4D7 and clone 5E4E11 (FIG. 12A); viii) parainfluenza virus 3 MAbcombination of clone 4G5(1)E2H9 and clone 1F6C8 (FIG. 12B); ix) pooledparainfluenza 1-3 MAbs as described above (FIG. 13A); and x) combinedmixture of i)-ix) (FIG. 13B).

In one embodiment, the present invention contemplates a method toperform LDFA comprising a virus-specific antibody. In one embodiment,the antibody comprises a monoclonal antibody. In one embodiment, thevirus-specific monoclonal antibody comprises a fluorescent label. In oneembodiment, the fluoresently labeled monoclonal antibody comprises Flu Amonoclonal antibody (i.e., for example, with a PE label). In oneembodiment, the fluorescently labeled monoclonal antibody comprises FluB monoclonal antibody (i.e., for example, with a FITC label). In oneembodiment, the fluorescently labeled monoclonal antibody comprises aRSV monoclonal antibody (i.e., for example, with a PE label). In oneembodiment, the fluorescently labeled monoclonal antibody comprises MPVmonoclonal antibody (i.e., for example, with a FITC label). In oneembodiment, the fluorescently labeled monoclonal antibody comprises aparainfluenza (i.e., for example, PIV-1, -2 and -3) monoclonal antibody(i.e., for example, with a PE label). In one embodiment, thefluorescently labeled monoclonal antibody comprises an adenvirusmonoclonal antibody (i.e., for example, with a FITC label).

In one embodiment, the present invention contemplates a method to detectat least eight (8) and identify at least five (5) viruses comprisingincubating a single liquid sample with at least one PE-labeledmonoclonal antibody directed to a first virus and at least oneFITC-labeled monoclonal antibody is directed to a second virus. In oneembodiment, a first aliquot of the liquid sample comprises a PE-labeledFlu A monoclonal antibody and a FITC-labeled Flu B monoclonal antibody.In one embodiment, a second aliquot of the liquid sample comprises aPE-labled RSV monoclonal antibody and a FITC-labeled MPV monoclonalantibody. In one embodiment, a third aliquot of the liquid samplecomprises a PE-labeled PIV monoclonal antibody and a FITC-labeledadenovirus monoclonal antibody. The present method has considerableadvantages over those DFAs currently available as this method can detectand identify at least eight (8) respiratory viruses using three (3)aliquots from a single biological sample.

a. Sapogenin Enhanced Methods

In one embodiment, the present invention contemplates a liquid directfluorescence assay to detect virus that do not require incubation ineither a fixative or a dehydration agent. These fixative and/ordehydration agents are required in DFAs because the virus-infected cellsare adhered to a glass substrate to facilitate microscopic viewing andexamiation. In one embodiment, the present method comprises unfixedcells, wherein the liquid does not contain fixatives or non-aqueoussolvents (i.e, for example, alcohols, acetone, aldehydes, toluene,etc.). In one embodiment, the invention contemplates a LDFA whereincells are permeabilized with a detergent agent. In one embodiment, thedetergent comprises sapogenin. Although it is not necessary tounderstand the mechanism of an invention, it is believed that adetergent agent provides improved cell permeability of fluorescentlylabeled antibodies in comparison to conventional fixatives anddehydration agents. It is further believed that this improvedfluorescently labeled antibody permeability results in greater bindingwith viral antigens, thereby resulting in improved signal strength. Itis further believed that the improved signal strength providesequivalent sensitivity and improved accuracy for the present LDFA versuscurrently available DFAs for virus detection and identification.

Saponins, including sapogenin, have been reported as a lipid-baseddetergent. Sapogenin has been suggested as being able to enhance thecontrast of cells and sub-cellular morphology in histological slidepreparations. Such histology preparations typically use dehydrationsolvents (i.e., for example, toluene) but may employ fluorescent labels.Sapogenin was not used to facilitate the detection of viruses (i.e., forexample, influenza). Farrell et al., “Biological Sample ProcessingComposition and Method,” United States Patent Publication No.2007/0172911 (herein incorporated by reference). Saponins have furtherbeen reported to permeabilize cell membranes. Saponin used inconjunction with Evan's blue and propidium iodide staining of influenzavirus was not observed to detect the virus in a solution based assay.Johansen et al., “Compositions and Methods for Treatment of ViralDiseases,” United States Patent Publication No. 2008/0161324 (hereinincorporated by reference).

Saponins have detergent-like properties and have been reported useful asfoaming agents. Further, saponins may be used as immunological adjuvantsfor viral vaccines including influenza and, when fluorescently labeled,is capable of detecting cell surface markers. Marciani et al.,“Triterpene Saponin Analogs Having Adjuvant and ImmunostimulatoryActivity,” U.S. Pat. No. 5,977,081 (1999); U.S. Pat. No. 6,262,029; andU.S. Pat. No. 6,080,725 (both herein incorporated by reference).Saponins may also be combined with nutraceuticals and/orpharmaceuticals. For example, saponins may suppress HIV replication.Dobbins et al., “Process For Isolating Saponins From Soybean-DerivedMaterials,” U.S. Pat. No. 6,355,816 (2002) (herein incorporated byreference).

In one embodiment, the present invention contemplates a method toperform a liquid direct flourescent assay (LDFA) comprising sapogenin.Although it is not necessary to understand the mechanism of aninvention, it is believed that sapogenin offers significant advantagesover currently known DFA methods because the compound permeabilizes thecells instead of fixing the cells. It is further believed thatpermeabilization has the advantages of: i) treating the infected cellswith a mild surfactant, thereby allowing the cells to maintain theirthree dimensional structure while being stained with a proteincounterstain and labeled antibodies; ii) solubilizing the lipid portionsof a cell membrane; and iii) allowing larger dye molecules andantibodies access to the cell's interior. In one embodiment, the presentinvention contemplates a method comprising LDFA, wherein sapogeneintreatment improves virus detection and identification by decreasingbackground noise and improving antibody signal strength.

III. Portable Fluorescent Reader Devices

Fluorescence microscopy has allowed the examination of fluorescentlystained specimens by visual inspection. However, automatingfluorescently labeled cell counts in conjunction with total cell countsprovides an opportunity for fast and reliable diagnostic information(i.e., for example, cytometers having internal alogrithms). In oneembodiment, the present invention contemplates a device that generatesdata that compare favorably with those from a conventionalhema-cytometer, yet it eliminates the variability associated withsubjective interpretation. In one embodiment, the device is capable ofdisplaying test results in less than one minute per sample. In oneembodiment, the device further automatically calculates cell viability.

In one embodiment, the device may be used together with a plurality ofstaining agents. In one embodiment, the staining agents provide fortesting a wide variety of nucleated cell lines, including, but notlimited to, mammalian cells, hybridomas and ficoll preparations. In oneembodiment, the staining agents are detected by a fluorescentmicroscopy-based imaging system that streamlines cell countingprocedures. For example, the staining agents may include, but are notlimited to, a plurality of fluorescently labeled monoclonal antibodiesand nucleic acid dyes. In one embodiment, the nucleic acid dyes includebut are not limited to, propidium iodide, acridine orange, or ethidiumbromide.

In one embodiment, the device comprises an epi-illumination microscopewhere a charged couple device collected emitted fluorescence thatresults from illumination by light emitting diodes. In one embodiment,the device comprises a sample drawer configured to accept a sample traycomprising a plurality of samples (i.e., for example, a multi-wellsample slide). In one embodiment, the illumination is accomplished byhigh intensity mercury-arc or quartz-halogen light emitting diodes.Following illumination and collection of the fluorescence, the cellcount is generated by image analysis using an internal algorithm. In oneembodiment, the device visually displays test results on a touch screen.In one embodiment, the device is capable of exporting the test resultsto an independent storage device (i.e., for example, a computer).

In one embodiment, the device is compatible a method comprising: a)pipeting a sample into at least one microwell of a multiwell microscopeslide; b) loading the slide onto a slide tray; and c) inserting theslide tray into the sample drawer of the device. In one embodiment, thesample comprises a cell suspension and a plurality of staining reagents.Total cells (live and dead) may be counted by staining with, forexample, by Thioflavin T, acridine orange, non-specific fluorescentdyes, or any particle attached to an antibody that is detectable by amicroscope. Ethidium bromide is further added to identify the deadcells, wherein the number of live cells is then determined bysubtraction.

While the present invention contemplates that many different devicesthat would be compatible with the presently contemplated method,preferred specifications may include, but are not limited to: i) samplevolume of approximately 8 μl sample; ii) dynamic range: 5×10⁴ to 1×10⁷cells/mL; iii) detectable cell diameter between approximately 8-40microns; iv) calculation software that determines the labeled cell countand % viability by counting labled cells and total cells in thespecified volume of the image fields; v) a fluorescence microscopehaving, for example, a charge coupled device camera; iv) two lightemitting diodes (LEDs) @ 470 & 530 nm respectively; v) total analysistime in approximately 1 minute per test; vi) processing of six (6)images/test; vi) approximate dimensions: 37.5 H×25 D×30 W cm vii)approximate weight: 9 kg (20 lbs); vii) optimal operating temperaturebetween approximately 10-35° C.; viii) optimal operating humiditybetween approximately 20-80% relative humidity; ix) optimal operatingaltitude of up to approximately 2,000 meters; and x) power requirements:100-240 VAC, 50-60 Hz. See, FIG. 14.

In one embodiment, the present invention contemplates a microscope slidecomprising a plurality of sample wells (˜200 μl). In one embodiment, themicrowell comprises an inlet port. In one embodiment, the microwellcomprises an outlet port. In one embodiment, the microwell comprises andinlet port and an outlet port. In one embodiment, the microwell iscovered by a coverslip. In one embodiment, the ports are compatible witha 10 μl pipet tip. See, FIG. 15.

In one embodiment, the first, second, third, and fourth aliquots areidependently placed on a glass substrate. In one embodiment, the glasssubstrate comprises at least four (4) sample wells, such that eachindependent sample is placed within a separate microwell. In oneembodiment, each microwell comprises a side inlet port and a side outletport. In one embodiment, the microwell comprises a permanent cover.

IV. Integration of Device & Method

In one embodiment, the present invention contemplates a methodcomprising: a) providing; i) a fluorescent cytometer compatible with asample container comprising a plurality of samples; ii) a biologicalspecimen comprising a plurality of cells; b) labeling the cells with aplurality of fluorescent dyes; c) placing the labeled cells within thesample container; and d) examining the sample container for fluorescenceusing the cytometer.

In one embodiment, the present invention contemplates a devicecomprising a microprocessor comprising an algorithm capable ofdifferentiating between a plurality of fluorescent signals. In oneembodiment, a first fluorescent signal comprises a PE signal. In oneembodiment, the PE signal appears as a golden-yellow fluorescent stain.In one embodiment, a second fluorescent signal comprises an FITC signal.In one embodiment, the FITC signal appears as an apple-green fluorescentstain. In one embodiment, a third fluorescent signal comprises apropidium iodide signal. In one embodiment, the propidium iodide signalappears as a red fluorescent stain.

In one embodiment, the rinsed and centrifuged liquid sample is loadedonto a sample container comprising a glass substrate. In one embodiment,the glass substrate comprises a plurality of independent samples. In oneembodiment, the glass substrate is compatible with a device comprisingan algorithm capable of detecting and evaluating a plurality offluorescent signals. In one embodiment, the device detects the signalsfrom each independent sample.

Although there are many different methods of preparing and examiningcells, the following protocol is described in detail as but one examplethat is compatible with the presently disclosed invention. Briefly, theprocessing of the specimen for reading in the instrument is as follows.A nasopharengeal (NP) swab or aspirated NP specimen is placed in atransport medium (i.e., for example, phosphate buffered saline; PBS). Analiquot (˜1 ml) is transferred to a centrifuge tube for pelleting. Thecell pellet is resuspended in about 0.1 ml of PBS, vortexed to dispersethe cells and then 25 uL of the suspension are transferred to 4 separatetubes, to which are added, respectively, 1 drop of fluorescein-labeled,Non-Immune mouse Ab, Flu A MAb, Flu B MAb and RSV MAb and allowed tostand at room temperature for 10 minutes. Optionally, each of these MAbsolutions also contains sapogenin as a cell permeabilization reagent,propidium iodide to counterstain the nuclei of all the virus infectedand uninfected NP cells. 1.5 ml of PBS is then added to each tube whichis centrifuged and the supernatant of each (which contains the excessMAb, counterstain and permeabilization reagent) is decanted. Each cellpellet is resuspended in a minimal volume of PBS. The Non-Immune mouseAb is included as a control since there are some specimens that containcells that bind the Fc portion of murine antibodies. In such cases, allwells that contain fluorescein labeled MAb will show fluorescence andthe state of infection of the specimen cannot be determined by thismethod. About 10 uL of each of the 4 suspensions are pipetted into eachof 4 wells of a special slide in the order listed above; the 4 separatewells are covered by a coverslip and each well has an entry port on eachside. Each well has a capacity of about 7 uL. The slide containing thecell suspensions is inserted into a slide tray of the cytometer devicewhich automatically moves the slide inside the instrument where itsalignment is first checked and then moved to successively position eachwell beneath a 5× objective. For example, the alignment may takeapproximately 95 sec. and each microwell may take approximately 2minutes (i.e., a total of 8 minuntes for four successive microwellreads). The instrument may contain at least 2 LED's. A first LED emitslight at a wavelength to excite fluorescein. A second LED that emitslight to excite the propidium iodide counterstain. There are narrow bandwavelength filters interposed between the emitted light and the CCD. Ateach well, a predetermined number of fields (9 or 16 out of a possible27) is excited and imaged separately (first the fluorescein immediatelyfollowed by the propidium iodide) at both LED wavelengths which arecaptured by the CCD. The algorithm is then used to analyze the images,identifying specific virus-infected cells by virtue of size and theco-location of the fluorescein-labeled MAb and propidium iodide andnon-infected cells by virtue of size and propidium iodide stain. Thealgorithm provides the number of infected cells and total number ofcells in the fields and wells examined. Upon completion of reading the 4wells, the slide is ejected from the instrument, ready for the nextspecimen-containing slide.

In one embodiment, the present invention contemplates detecting andidentifying a virus using mixtures of publicly available MAbs. In oneembodiment, each virus may be detected using at least one labeled MAb.See Table 9.

TABLE 9 Representative MAb Clones For Virus Identification VirusSpecificity Clonal Designation Fluorescent Label Source Influenza AVirus 2H3C5 PE Diagnostic Hybrids, Inc. Athens, OH; A(6)B11 Cat. No.01-013102.v2 Influenza B Virus 8C7E11 FITC Diagnostic Hybrids, Inc.Athens, OH; 9B4D9 Cat. No. 01-013202.v2 Respiratory Syncytial Virus3A4D9 PE Diagnostic Hybrids, Inc Athens, OH; 4F9G3 Cat. No. 01-013302.v2Metapneumovirus Clone #4 FITC US Patent Application Publication C2C10Number 2007/0248962, herein incorporated by reference MetapneumovirusClone #23 FITC US Patent Application Publication C2D11 Number2007/0248962, herein incorporated by reference Metapneumovirus Clone #28FITC US Patent Application Publication T3H11 Number 2007/0248962, hereinincorporated by reference Parainfluenza 1 Virus 1D8E10 PE DiagnosticHybrids, Inc Athens, OH; 9F61C9 Cat. No. 01-013502.v2 Parainfluenza 2Virus 2E4D7 PE Diagnostic Hybrids, Inc Athens, OH; 5E4E11 Cat. No.01-013602.v2 Parainfluenza 3 Virus 4G5(1)E2H9 PE Diagnostic Hybrids, IncAthens, OH; 1F6C8 Cat. No. 01-013702.v2 Adenovirus 8H2C9 FITC DiagnosticHybrids, Inc. Athens, OH; 2H10E2 Cat. No. 01-013402.v2 4H6C9

In one embodiment, an influenza A reagent comprises at least onePE-labeled MAb selected from the group comprising clone 2H3C5 or cloneA(6)B11. In one embodiment, an influenza B reagent comprises at leastone FITC-labeled MAb selected from the group comprising clone 8C7E11 orclone 9B4D9. In one embodiment, a respiratory syncytial virus reagentcomprises at least one PE-labeled MAb selected from the group comprisingclone 3A4D9 or clone 4F9G3. In one embodiment, a metapneumovirus reagentcomprises at least one FITC-labeled MAb selected from the groupcomprising clone #4, clone #23, or clone #28. In one embodiment, aparainfluenza 1 reagent comprises at least one PE-labeled MAb selectedfrom the group comprising clone 1D8E10 or clone 9F61C9. In oneembodiment, a parainfluenza 2 reagent comprises at least one PE-labeledMAb selected from the group comprising clone 4G5(1)E2H9 or clone 1F6C8.In one embodiment, a parainfluenza 3 reagent comprises at least onePE-labeled MAb selected from the group comprising clone 4G5(1)E2H9 orclone 1F6C8. In one embodiment, an adenovirus reagent comprises at leastone FITC-labeled MAb selected from the group comprising clone 8H2C9,clone 2H10E2, or clone 4H6C9.

Although there are many different methods of detecting and identifyingviral infected cells, the following protocol is described in detail asbut one example that is compatible with the presently disclosedinvention. In one embodiment, a specimen prepared as described above isaliquoted into three (3) independent wells on a glass substrate (i.e.,for example, a microscope slice). In one embodiment, the method furthercomprises contacting an influenza A reagent and an influenza B reagentwith the sample in a first well. In one embodiment, the method furthercomprises contacting a respiratory syncytial virus reagent and ametapnuemovirus reagent with the sample in a second well. In oneembodiment, a third well comprises a parainfluenza 1 reagent, aparainfluenza 2 reagent, a parainfluenza 3 reagent and an adenovirusreagent. In one embodiment, the method further comprises detectinginfluenza A in the first well upon appearance of a golden-yellowfluorescent stain. In one embodiment, the method further comprisesdetecting the absence of influenza A in the first well upon appearanceof only a red stain. In one embodiment, the method further comprisesdetecting influenza B in the first well upon appearance of anapple-green fluorescent stain. In one embodiment, the method furthercomprisies detecting the absence of influenza B in the first well uponappearance of only a red stain. In one embodiment, the method furthercomprises detecting respiratory syncytial virus in the second well uponappearance of a golden-yellow fluorescent stain. In one embodiment, themethod further compries detecting the absence of respiratory syncytialvirus in the second well upon appearance of only a red stain. In oneembodiment, the method further comprises detecting metapneumovirus inthe second well upon appearance of an apple-green fluorescent stain. Inone embodiment, the method further comprises detecting the absence ofmetapneumovirus in the second well upon appearance of only a red stain.In one embodiment, the method further comprises detecting at least oneparainfluenza virus in the third well upon appearance of a golden-yellowfluorescent stain. In one embodiment, the at least one parainfluenzavirus is selected from the group comprising parainfluenza 1,parainfluenza 2, or parainfluenza 3. In one embodiment, the methodfurther comprises detecting the absence of any parainfluenza virus inthe third well upon appearance of only a red stain. In one embodiment,the method further comprises detecting an adenovirus in the third wellupon appearance of an apple-green fluorescent stain. In one embodiment,the method further comprises detecting the absence of an adenovirus inthe third well upon appearance of only a red stain.

V. Antibodies

The present invention provides isolated antibodies (i.e., for example,polyclonal or monoclonal). In one embodiment, the present inventionprovides monoclonal antibodies that specifically bind to viral epitopescomprised of at least five amino acid residues or lipid residue. Theseantibodies find use in the detection methods described above.

An antibody against a viral epitope of the present invention may be anymonoclonal or polyclonal antibody, as long as it can recognize theepitope. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animalwhose antibody titer has been confirmed (e.g., a mouse) is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter and antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Various methods may be used for screening for a hybridoma producing theantibody (e.g., against a viral epitope of the present invention). Forexample, where a supernatant of the hybridoma is added to a solid phase(e.g., microplate) to which antibody is adsorbed directly or togetherwith a carrier and then an anti-immunoglobulin antibody (if mouse cellsare used in cell fusion, anti-mouse immunoglobulin antibody is used) orProtein A labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase. Alternately, a supernatant of the hybridoma is added to a solidphase to which an anti-immunoglobulin antibody or Protein A is adsorbedand then the protein labeled with a radioactive substance or an enzymeis added to detect the monoclonal antibody against the protein bound tothe solid phase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification. Normally, a medium for animal cells towhich HAT (hypoxanthine, aminopterin, thymidine) are added is employed.Any selection and growth medium can be employed as long as the hybridomacan grow. For example, RPMI 1640 medium containing 1% to 20%, preferably10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetalbovine serum, a serum free medium for cultivation of a hybridoma(SFM-101, Nissui Seiyaku) and the like can be used. Normally, thecultivation is carried out at 20° C. to 40° C., preferably 37° C. forabout 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO₂gas. The antibody titer of the supernatant of a hybridoma culture can bemeasured according to the same manner as described above with respect tothe antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody (e.g., against aviral epitope of the present invention) can be carried out according tothe same manner as those of conventional polyclonal antibodies such asseparation and purification of immunoglobulins, for example,salting-out, alcoholic precipitation, isoelectric point precipitation,electrophoresis, adsorption and desorption with ion exchangers (e.g.,DEAE), ultracentrifugation, gel filtration, or a specific purificationmethod wherein only an antibody is collected with an active adsorbentsuch as an antigen-binding solid phase, Protein A or Protein G anddissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. A material containing theantibody against is recovered from the immunized animal and the antibodyis separated and purified.

As to the complex of the immunogen and the carrier protein to be usedfor immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to a hapten in a weight ratio of about 0.1 parts to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

In addition, various condensing agents can be used for coupling of ahapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like,of an animal immunized by the above method. The antibody titer in theantiserum can be measured according to the same manner as that describedabove with respect to the supernatant of the hybridoma culture.Separation and purification of the antibody can be carried out accordingto the same separation and purification method of immunoglobulin as thatdescribed with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, a protein expressed resultingfrom a virus infection (further including a gene having a nucleotidesequence partly altered) can be used as the immunogen. Further,fragments of the protein may be used. Fragments may be obtained by anymethods including, but not limited to expressing a fragment of the gene,enzymatic processing of the protein, chemical synthesis, and the like.

The present invention may be practiced using any antibody. As describedabove, preferred antibodies comprise monoclonal antibodies that areproduce from hybridoma cell cultures. In one embodiment, the presentinvention contemplates a hybridoma cell culture that produces amonoclonal antibody, wherein said monoclonal antibody has specificaffinity for a viral antigen derived from a virus selected from thegroup including, but not limited to, influenza A, influenza B,adenovirus, parainfluenza 1, parainfluenza 2, parainfluenza 3,parainfluenza 4, respiratory syncytial virus, human metapneumovirus,varicella zoster virus, herpes simplex virus-1, herpes simplex virus-2,cytomegalovirus IE, coronavirus 229E, coronavirus 0C43, severe acuterespiratory syndrome virus, coxsackie virus B3 VP1 Pan-EV, Poliovirus 1VP1 Pan-EV, enterovirus 70 specific, enterovirus 71 specific,enterovirus 71/Coxsackie A16 bispecific, bocavirus, and human papillomavirus. In one embodiment, the present invention contemplates a hybridomacell culture that produces a monoclonal antibody, wherein the monoclonalantibody has specific affinity for a bacterial antigen derived from abacteria selected from the group including, but not limited to,chlamydia, methicillin resistant Staphylococcus aureus, Group AStreptococcus, and Group B Streptococcus. In one embodiment, the presentinvention contemplates a hybridoma cell culture that produces amonoclonal antibody, wherein the monoclonal antibody has specificaffinity for a small organic molecule selected from the group including,but not limited to, nicotine or cotinine

A. Influenza A/Respiratory Virus Monoclonal Antibodies

In one embodiment, the present invention contemplates a specificmonoclonal antibody capable of qualitatively detecting and identifyinginfluenza A viral antigens. In one embodiment, the present inventioncontemplates a specific monoclonal antibody capable of screening forviral antigens selected from the group comprising influenza B virusantigens, respiratory syncytial virus antigens, adenovirus antigens, andparainfluenza virus types 1, 2, and 3 antigens. In one embodiment, thedetecting and/or screening comprises directly testing cells derived fromrespiratory biological specimens. In one embodiment, the detectingand/or screening comprises a method performed in a cell culture byimmunofluorescence using the monoclonal antibodies (MAbs).

In one embodiment, the MAbs are provided in a kit comprising a pluralityof viral antigen-specific murine MAbs. In one embodiment, MAbs forinfluenza A virus are directly labeled with R-phycoerythrin (i.e., forexample, emitting a golden-yellow fluorescence). In one embodiment, MAbsfor influenza B virus, respiratory syncytial virus, adenovirus, andparainfluenza virus types 1, 2, and 3, are directly labeled withfluorescein isothiocyanate (i.e., for example, emitting an apple-greenfluorescence). Although it is not necessary to understand the mechanismof an invention, it is believed that these MAbs result in thequalitative and quantitative detection of these viruses.

In one embodiment, the present invention contemplates a methodcomprising isolating cells derived from a clinical and/or biologicalspecimen, or from a cell culture. In one embodiment, the cells areprocessed, stained and labeled. In one embodiment, the labeling resultsin a golden-yellow fluorescence from an Influenza A virus infected cell.In one embodiment, the labeling results in an apple-green fluorescencefrom an influenza B virus, respiratory syncytial virus, adenovirus, orparainfluenza virus types (1-3) infected cell. 1. Hybridoma Development

In one embodiment, the present invention contemplates a compositioncomprising an MAb 2H3C5. In one embodiment, the composition may furthercomprise an MAb 10B12C11. In one embodiment, the composition may furthercomprise an MAb A(6)B11. In one embodiment, the MAbs may be produced inmammalian hybridomas including, but not limited to, murine hybridomas.See, Table 10.

TABLE 10 Representative Hybridoma Clones For Influenza A/RespiratoryVirus MAbs Antigen for Animal for Target MAb Clone ID immunizationsimmunizations protein Influenza A virus Influenza A virus (Texas BALB/cmice Unknown¹ 2H3C5 1/77, H3N2), purified from amniotic fluids InfluenzaA virus Unknown, the hybridoma Unknown Unknown^(a) A(6)B11 purchasedfrom ZymeTx, Inc² ¹Target protein denaturation by the sample process forWestern blotting precludes the target protein identification. ²OklahomaCity, OK. ^(a)As disclosed herein.

Although it is not necessary to understand the mechanism of aninvention, it is believed that an MAbs having the highest antigenaffinity would give the brightest fluorescent staining In oneembodiment, the present invention contemplates a method comprisingscreening MAb producing hybridomas with high affinity MAbs usingindirect fluorescent assay (IFA) on infected cell cultures. For example,influenza A viruses were inoculated onto R-Mix® (Diagnostic Hybrids,Inc., Athens, Ohio) cell monolayers in 96-well plates and grown for 24hours at 35° to 37° C. The cells were then fixed with acetone, washedand incubated at 35° to 37° C. with hybridoma cell supernatant for 30minutes in a humidified incubator. The cells were again washed and thenincubated at 35° to 37° C. in a humidified incubator with FITC-labeledgoat anti-mouse antibody for 30 minutes. The resulting stains were usedto choose the best clones to take forward to the next step in thedevelopment process (i.e., for example, small scale purification anddirect labeling).

Hybridomas that were screened and selected in this manner resulted inthe identification of specific isotypes. For example, one immunogen thatwas used for mouse immunization was influenza A antigen (Texas 1/77,H3N2), purified from a commercially available amniotic fluid (R02302;Biodesign). See, Table 11.

TABLE 11 Influenza A Hybridoma Product Candidates Fluorescence Clonename Intensity* Isotype A(6)B11 ++++ IgG2a (k) 2H3C5 ++++ IgG2b (k)10B12C11 +++++ IgG1 (k) *Subjective observed fluorescence intensity: + =weak and ++++++ = brightest.

2. Monoclonal Antibody Purification

Hybridoma monoclonal antibodies as produced above were subsequentlypurified from cell culture supernatant by Protein G affinity using FastProtein Liquid Chromatography (FPLC). MAb purity was checked by SDS-PAGEwherein an internal quality control standard ensured a minimum purity ofat least 90%.

The resultant purified MAbs were further isolated on a 4%→20% gradientSDS-PAGE electropherogram gel under denaturing conditions. FIG. 6. Thepurity of each of the MAbs was determined by scanning densitometry. See,Table 12.

TABLE 12 Purity Determination Of Representative mAbs. Lane MAb PurityAntibody Clone Lot # Position (%) Influenza A virus 2H3C5 031806 C1 100072506A C2 99.9 072506B C3 99.8 Influenza A virus A(6)B11 040506 A2 99.8080505-2FA C5 100 082106A C6 100

The data show that the purity of each representative MAb exceeded theminimal quality control 90% purity requirement, wherein the purity forall the MAbs ranges between approximately 99.7 to 100%.

3. Monoclonal Antibody Binding Affinities

The relative affinities of MAbs for various viral antigens weredetermined by ELISA assay as follows:

-   -   i) Lysates of a virus-infected cell (i.e., for example,        influenza A (Texas/1/77 H3N2)) were obtained from Biodesign        International were used to coat 96-well microtiter plates.    -   ii) Two-fold serial dilutions of the MAbs were incubated on each        plate.    -   iii) The binding of each MAb to the immobilized viral antigen        was detected by using a goat anti-mouse IgG antibody, conjugated        to horseradish peroxidase (HRP).        Results for an assay of: i) MAb (10B12C11); ii) MAbs 2H3C5        and; iii) MAb A(6)B11) to influenza A demonstrate binding        affinities corresponding to kDa values of ˜0.013 nM for 2H3C5,        ˜0.36 nM for 10B12C11, and ˜0.96 nM for A(6)B11. The binding        affinity for influenza A virus MAb 2H3C5 was 2-10 orders of        magnitude higher than the 10B12C11 MAb and/or the A(6)B11 MAbs.        FIG. 7. In one embodiment, the 2H3C5 MAb comprises a specific        affinity for influenza A antigen.

4. Monoclonal Antibody Characterization

A variety of methods were used to characterize influenza A virus MAbs inthe present invention. See, Table 13.

TABLE 13 Characterization Assays for the Representative MAbs FPLCWestern In Situ Staining In Situ Staining Virus Target Clone PurityELISA blotting (lab strains) (clinical isolates) Influenza A 2H3C5 YesYes  Negative³ Positive Positive Influenza A A(6)B11 Yes Yes NegativePositive Positive ³Negative result due to the epitope specimen treatmentdenaturing effectsThe data show that 2H3C5 and A(6)B11 were both capable of detectinginfluenza A.

a. Analytical Sensitivity

Analytical sensitivity of representative MAbs were evaluated usinginfluenza A virus. For example, strain Victoria (H3N2; ATCC VR-822) wasused. In this determination, two 96-well cell culture plates wereinoculated with the influenza A virus diluted to a level of 1.0 50%Tissue Culture Infectious Dose (1.0 TCID₅₀) per 0.2-mL inoculum. Theplates were incubated at 35° to 37° C. for 24-hours and then stained.The assay was performed four times. An average of 35 positive wells (outof 96) detected with a combination of a MAb 2H3C5 and MAb A(6)B11.Likewise, an average of 35 positive wells (out of 96) was detected witha combination of MAb 10B12C11 and Mab A(6)B11. See, Table 14.

TABLE 14 Analytical Sensitivity of MAb Combinations To Influenza AVirus. Positive wells Mean ± SD 2H3C5/ 10B12C11/ 2H3C5/ 10B12C11/ TestNumber A(6)B11 A(6)B11 A(6)B11 A(6)B11 1 23 26 34.3 ± 12.0 34.8 ± 9.7 226 27 3 39 44 4 49 42 Paired t-test = 0.86

The data show that at 1.0 TCID₅₀, both MAb combinations positivelyidentified influenza A virus infected cells.

b. Detection Limits

The analytical detection limits were determined for each MAbcombination. Using the 2H3C5/A(6)B11 MAb combination as an example, theassay conditions were similar to those described above, with resultsreported in a different manner (numbers of fluorescent cells per cellmonolayer). For example, influenza A virus (Victoria) stock viruspreparation was diluted to a value of 359 TCID₅₀ per inoculum, andserial 2-fold dilutions were then made to a final calculated value of0.7 TCID₅₀. Each dilution of virus was inoculated into six confluentmonolayers of R-Mix® cells in shell vials, centrifuged at 700×g for 60minutes and incubated at 35° to 37° C. for 48 hours.

The 2H3C5/A(6)B11 MAb combination or the 10B12C11/A(6)B11 MAbcombination was used to stain 3 shell vials of each viral dilution of a96-well plate. The determinations were performed in triplicate and thenumber of positive cells per well was counted. Fluorescent cells werecounted on each coverslip at the indicated virus dilutions.

TABLE 15 Analytical Detection Limits of Representative MAbs forinfluenza A virus (Victoria). Influenza A virus Fluorescent stainingcells/cell (Victoria) monolayer (triplicate samplings) TCID₅₀ perinoculum 2H3C5/A(6)B11 10B12C11/A(6)B11 5.60 2, 1, 0 3, 1, 0 2.80 1, 0,2 1, 0, 1 1.40 0, 1, 2 0, 0, 1 0.70 0, 0, 0 0, 0, 0

The data show that both fluorescent antibody stain combinationsperformed to comparable limits, with a minimum viral dilution detectedbetween 1.4 and 0.7 TCID₅₀.

5. Performance of Viral Monoclonal Antibodies

In one embodiment, the present invention contemplates a viral monoclonalantibody labeled with a fluorescent moiety including, but not limitedto, FITC or R-PE. In one embodiment, a fluorescein-labeled MAb exhibitsa fluorescent apple-green color. In one embodiment, aphycoerythrin-labeled MAb exhibits a fluorescent golden-yellow color.Although it is not necessary to understand the mechanism of aninvention, it is believed that when viewed through a microscope fittedwith standard FITC filters; both fluorescent colors may be visualizedusing the same FITC-filter set on a fluorescence microscope.

In one embodiment, a first MAb having specificity for influenza A virusis labeled with R-PE (golden-yellow) and a second MAb having specificityfor influenza B virus, respiratory syncytial virus, adenovirus,parainfluenza viruses types 1, 2, and 3 is labeled with FITC(apple-green). In one embodiment, the present invention contemplates afirst DFA kit capable of differentiating between influenza A virus andrespiratory virus, wherein cells infected by the influenza A virus staingolden-yellow. FIG. 8A. In one embodiment, the present inventioncontemplates a second DFA kit capable of differentiating between ainfluenza A virus focus and respiratory virus focus, wherein cellsinfected by the influenza virus stain apple-green FIG. 8B. In either thefirst or second DFA kit cells infected with influenza B virus,respiratory syncytial virus, adenovirus, and parainfluenza virus types1, 2, and 3 infected cell cultures may also stain apple-green. In oneembodiment, the influenza A virus MAb has specificity to a plurality ofinfluenza A strains. Although it is not necessary to understand themechanism of an invention, it is believed that a fluorescent stainingvirus focus is either one cell or a group of closely adjacent cells thatfluoresce when stained using fluorescently labeled-specific antibodies.It is further believed that viruses including, but not limited to,influenza A, influenza B, and adenovirus produce only one or a fewfluorescent staining cells per viable infectious virus.

6. Cross Reactivity Testing

The 2H3C5/A(6)B11MAb combination was evaluated for cross reactivityagainst a number of microorganisms (i.e., for example, viruses and/orbacteria) that could be encountered during testing for respiratoryviruses either as an infectious organism or a contaminant.

Stringent conditions for cross-reactivity testing were achieved by usinga high concentration of MAbs and high titers of microorganisms.Depending on the particular virus, 71-1,400 TCID₅₀ per inoculum wereused for testing. Bacteria at Colony Forming Units (CFUs) ranging from6.4×10⁴ to 2.93×10⁷/10 μL were tested.

Conjugated MAbs were used at a higher concentration (i.e., for example,1.5×) than used in clinical testing regimens, but were low enough to beable to distinguish “signal” from the general background. With the 1.5×concentration, the specific infected targets exhibited equally “bright”targets as the 1× concentration (i.e., for example, there was noquenching observed at higher concentrations) although there was somebackground nonspecific “glow”.

Some microorganisms were commercially purchased, e.g., American TypeCulture Collection. Sixty-six (66) virus strains, 17 host culture celltypes, 25 bacteria, three bacterial Chlamydia sp., one yeast and oneprotozoa cultures were examined for specificity and cross-reactivity,including Staphylococcus aureus (Cowan strain), a known protein Aproducing bacterium. These microorganisms were cultured in accordancewith the recommended protocols, and frozen stocks were prepared.

Amounts of microorganisms were selected in order to ensure that afluorescence signal would be easily detected by examination using afluorescence microscope. Depending on the particular virus, 71-1,400TCID₅₀ viral inoculum was inoculated into shell vial or multi-well platecell cultures and incubated for 24 to 48 hours, to yield a 1+ to 3+cytopathic effect (CPE), processed and stained with the 1.5× testreagent. Stained cells were examined at 200× magnification. Bacteriawere cultured, processed as suspensions, then spotted on microscopeslides at CFUs ranging from 6.4×10⁴ to 2.93×10⁷/well in a 10 μL dot andthen stained with the 1.5×MAbs preparation. Stained slides were examinedat 400× magnification. Some microorganisms were procured from anexternal source as prepared microscope slides, intended to be used aspositive controls for assays. Cell cultures were tested as intactmonolayers or acetone-fixed cell spots. Cell lines tested were thosenormally used to recover respiratory viruses.

For each of the virus strains tested, there was no cross reactivityobserved with the subject reagent. Each of the DFA reagent positivecontrols, showed bright fluorescence indicating a positive result whilethe test reagents showed only the red Evans Blue counterstain with novisible fluorescence. None of the uninfected cell culture lines show anyfluorescence or significant background staining Results of the2H3C5/A(6)B11MAb combination for viral cross-reactivity testing aresummarized. Table 16.

TABLE 16 Viral Cross Reactivity and Specificity Testing Labeled2H3C5/A(6)B11MAb TCID₅₀/Source/ Organism Strain or Type Lot NumberCombination or CFU Cell Line A-549 C560921 − monolayer Cell Line VeroC840914S − monolayer Cell Line HEp-2 C570914 − monolayer Cell Line MRC-5C510920 − monolayer Cell Line Mv1Lu C580915 − monolayer Cell Line MDCKC830921S − monolayer Cell Line pRK 480909 − cell spot Cell Line pCMKA470907 − cell spot Cell Line pRhMK CA490922 − cell spot Cell Line RhMKII A490909YS − cell spot Cell Line R-mix C960922 − monolayer Cell LineLLC-MK2 C860928 − monolayer Cell Line BGMK C530914 − monolayer Cell LineMRHF C440912 − monolayer Cell Line WI-38 850913 − cell spot Cell LineNCI-H292 C590929 − monolayer Cell Line RD C760908 − monolayerGolden-yellow Apple-green Adenovirus Type 1, VR-1 061704J − + 1,400Adenovirus Type 3, VR-3 112701A − + 1,400 Adenovirus Type 5, VR-5 070505− + 1,400 Adenovirus Type 6, VR-6 111201A − + 1,400 Adenovirus Type 7,VR-7 112701C − + 1,400 Adenovirus Type 10, VR-1087 111201B − + 1,400Adenovirus Type 13, VR-14 112701E − + 1,400 Adenovirus Type 14, VR-15033104 − + 1,400 Adenovirus Type 18, VR-19 011702A − + 1,400 AdenovirusType 31, VR-1109 011702B − + 1,400 Adenovirus Type 40, VR-931 012802 − +1,400 Adenovirus Type 41, VR-930 012802A − + 1,400 Influenza A Aichi,VR-547 (H3N2) 061704O + − 1,400 Influenza A Mal, VR-98 (H1N1) 061704D +− 1,400 Influenza A Hong Kong, VR-544 (H3N2) 040104 + − 1,400 InfluenzaA Denver, VR-546 (H1N1) 061704P + − 1,400 Influenza A Port Chalmers,VR-810 (H3N2) 061704C + − 1,400 Influenza A Victoria, VR-822 (H3N2)080204 + − 1,400 Influenza A New Jersey, VR-897 (H1N1) 110404 + − 1,400Influenza A WS, VR-1520 (H1N1) 061704B + − 1,400 Influenza A PR, VR-95(H1N1) 061704Q + − 1,400 Influenza B Hong Kong, VR-823 093004B − + 1,400Influenza B Maryland, VR-296 041105 − + 1,400 Influenza B Mass, VR-523093004A − + 1,400 Influenza B GL, VR-103 061704F − + 1,400 Influenza BTaiwan, VR-295 061704E − + 1,400 Influenza B JH-001 Isolate 061704R − +1,400 Influenza B Russia, VR-790 041105 − + 1,400 RSV Long, VR-26 GroupA 042204L − + 1,400 RSV Wash, VR-1401 Group B 042204W − + 1,400 RSV9320, VR-955 Group B 061704I − + 1,400 Parainfluenza 1 C-35, VR-94061704L − + 1,400 Parainfluenza 2 Greer, VR-92 061704M − + 1,400Parainfluenza 3 C 243, VR-93 061704N − + 1,400 Parainfluenza 4a M-25,VR-1378 112701U − 1,400 Parainfluenza 4b CH19503, VR-377 112701V − 1,400Metapneumovirus Subgroup A1 110905 − 1,400 Metapneumovirus Subgroup A2110805 − 1,400 Metapneumovirus Subgroup B1 111105 − 1,400Metapneumovirus Subgroup B2 110405 − 1,400 Coronavirus OC43, VR-1558041204A − 1,400 Coronavirus 229E, VR-740 121903 − 1,400 HSV-1 1F, VR-733052405 − 71 HSV-1 MacIntyre, VR-539 071005 − 71 HSV 2 MS, VR-540 112701Y− 71 HSV 2 Strain G, VR-734 052605 − 71 CMV Towne, VR-977 011503 − 430CMV Davis, VR-807 062005 − 430 CMV AD169, VR-538 052705 − 430Varicella-zoster Webster, VR-916 040504 − 430 Varicella-zoster Ellen,VR-1367 050903 − 430 Echovirus 4, Bion QEC-0008 − Control slideEchovirus 6, Bion QEC-0008 − Control slide Echovirus 9, Bion QEC-0008 −Control slide Echovirus 11, Bion QEC-0008 − Control slide Echovirus 30,Bion QEC-0008 − Control slide Echovirus 34, Bion QEC-0008 − Controlslide Coxsackievirus B1, Bion QCB-0011 − Control slide CoxsackievirusB2, Bion QCB-0011 − Control slide Coxsackievirus B3, Bion QCB-0011 −Control slide Coxsackievirus B4, Bion QCB-0011 − Control slideCoxsackievirus B5, Bion QCB-0011 − Control slide Coxsackievirus B6, BionQCB-0011 − Control slide Mumps Bion (CDC V5-004) QMU-0308 − Controlslide Rubeola (Measles) Bion QME-0424 − Control slide Rhinovirus 39 209Picornavirus, VR-340 112701EE − 1,400 Bacteria Acholeplasma laidlawi031404 − ~1.0 × 107 CFU  Bacteria Acinetobacter calcoaceticus 934332 −9.7 × 105 CFU Bacteria Bordetella bronchiseptica 031404 − 1.8 × 105 CFUBacteria Bordetella pertussis 031404 − 4.7 × 106 CFU BacteriaCorynebacterium diphtheriae 031404 − 2.5 × 106 CFU Bacteria Escherichiacoli 335472 − 2.6 × 105 CFU Bacteria Gardnerella vaginalis 3457511 − 5.0× 105 CFU Bacteria Haemophilis influenzae type A 031404 − 9.3 × 105 CFUBacteria Klebsiella pneumoniae 031404 − 6.4 × 106 CFU BacteriaLegionella pneumophila 031404 − 6.5 × 104 CFU Bacteria Moraxellacartarrhalis 031404 − 6.4 × 104 CFU Bacteria Mycoplasma hominis 031404 −~1.0 × 104 CFU  Bacteria Mycoplasma orale 031404 − ~1.0 × 104 CFU Bacteria Mycoplasma pneumoniae 031404 − ~1.0 × 104 CFU  BacteriaMycoplasma salivarium 031404 − ~1.0 × 107 CFU  Bacteria Neisseriagonorrhoeae 060805 − 1.3 × 106 CFU Bacteria Proteus mirabilis 440498 −2.1 × 106 CFU Bacteria Pseudomonas aeruginosa 031404 − 1.0 × 107 CFUBacteria Salmonella enteriditis 3457511 − 2.5 × 106 CFU BacteriaSalmonella typhimurium 363162 − 1.8 × 106 CFU Bacteria Staphylococcusaureus 081100 + 1.0 × 107 CFU Bacteria Streptococcus agalactiae 370784 −9.6 × 106 CFU Bacteria Streptococcus pneumoniae 031404 − 8.0 × 105 CFUBacteria Streptococcus pyogenes 031404 − 2.9 × 107 CFU BacteriaUreaplasma urealyticum 031404 − ~1.0 × 104 CFU  Chlamydia sp.Chlamydophila pneumoniae CP-0176 − Control slide Chlamydia sp.Chlamydophila psittaci FP-12- − Control slide 050218 Chlamydia sp.Chlamydia trachomatis 052705 − Control slide Yeast Candida glabrata992206 − 8.7 × 106 CFU Protozoa Trichomonas vaginalis 410721 − Controlslide

The 2H3C5/A(6)B11 MAb combination was found to be reactive with viraltarget-specific infected cells. Reactivity with Staphylococcus aureus ismost probably due to specific binding of the MAbs by the Protein Aproduced by Staphylococcus aureus. No reactivity was noted for all othermicroorganisms tested or for uninfected cells, as evidenced by nopositive fluorescent cells or elevated background fluorescence.

Staining of Staphylococcus aureus appear as small points of fluorescencewhile all other cultures were negative. Although it is not necessary tounderstand the mechanism of an invention, it is believed that Protein Aproduced by S. aureus may bind the Fc portion of somefluorescein-labeled monoclonal antibodies. It is further believed thatsuch binding can be distinguished from viral antigen binding on thebasis of morphology (i.e. for example, S. aureus-bound fluorescenceappears as small (˜1 micron diameter), bright dots). Consequently, falsepositives may be present in cell cultures with bacterial contamination.

The plates inoculated for the bacteria CFU confirmation yielded thefollowing results. The information is presented as CFU per mL and0.01-mL is used to dot each slide well that the reagent is tested. Theresults for the commercially tested slides as well as the mycoplasmatesting is listed and summarized. Table 17.

TABLE 17 Microorganism Cross-Reactivity And Specificity Testing ColoniesDilution CFU/ CFU/ Bacteria Counted Counted mL well Bordetellabronchiseptica 175 10⁻⁵ 1.75e7 1.75e5 Bordetella pertussis 465 10⁻⁶4.65e8 4.65e6 Legionella pneumophila 65 10⁻⁵ 6.50e6 6.50e4Corynebacterium diphtheriae 250 10⁻⁶ 2.50e8 2.50e6 Klebsiella pneumoniae64 10⁻⁷ 6.40e8 6.40e6 Streptococcus agalactiae 96 10⁻⁷ 9.60e8 9.60e6Haemophilis influenzae type A 93 10⁻⁶ 9.30e7 9.30e5 Pseudomonasaeruginosa 100 10⁻⁷ 1.00e9 1.00e7 Streptococcus pneumoniae 80 10⁻⁶8.00e7 8.00e5 Streptococcus pyogenes 293 10⁻⁷ 2.93e9 2.93e7 Moraxellacartarrhalis 64 10⁻⁵ 6.40e6 6.40e4 Staphylococcus aureus 104 10⁻⁷ 1.04e91.04e7 Neisseria gonorrhoeae 133 10⁻⁶ 1.33e8 1.33e6 Proteus mirabilis212 10⁻⁶ 2.12e8 2.12e6 Acinetobacter calcoaceticus 97 10⁻⁶ 9.70e7 9.70e5Escherichia coli 26 10⁻⁶  2.6e7 2.60e5 Gardnerella vaginalis 50 10⁻⁶5.00e7 5.00e5 Salmonella enteriditis 250 10⁻⁶ 2.50e8 2.50e6 Salmonellatyphimurium 177 10⁻⁶ 1.77e8 1.77e6 Candida glabrata 87 10⁻⁷ 8.70e8 8.7e6 Last Dilution with Visible Colonies Mycoplasma hominis 10⁻⁶Mycoplasma orale 10⁻⁶ Mycoplasma pneumoniae 10⁻⁶ Mycoplasma salivarium10⁻⁹ Ureaplasma urealyticum 10⁻⁶ Acholeplasma laidlawii 10⁻⁹ Chlamydiatrachomatis All tested on commercially available Chlamydia psittaciantigen control slides Trichomonas vaginalis Chlamydia pneumoniae

For each of the bacteria tested, there was no fluorescence observed at200 or 400× magnification with the subject reagent. The Staphylococcusaureus exhibits some slight fluorescence but that is expected due toProtein A binding of the MAb.

7. Stability Studies

The shelf life of the 2H3C5/A(6)B11MAb combination has been establishedas at least 12 months. Stability studies are conducted by storing theMAb combination at a temperature ranging between approximately 2° to 8°C. Various virus-infected R-Mix® cells cultured with human respiratoryviruses: Table 18.

TABLE 18 Virus strains used for Stability Studies Virus Source OtherIdentification Influenza A ATCC VR-822 Victoria (H3N2) Influenza B ATCCVR-295 Taiwan/2/62 Respiratory Syncytial Virus ATCC VR-1401 RSV-B, WashAdenovirus Type 1 ATCC VR-1 Adenovirus Type 14 ATCC VR-15 Parainfluenza1 ATCC VR-94 C-35 Parainfluenza 2 ATCC VR-92 Greer Parainfluenza 3 ATCCVR-93 C-243

Performance testing occurred at various time intervals during storagewherein characteristics were monitored including, but not limited to,performance, pH, color, and clarity. Each assay was run with dilutionseries of each of the MAb Conjugates at “neat” and a 1/16 dilution, then½ dilutions to as far as 1/256. Acceptance criterion is “brightfluorescence” observed in fixed, stained, infected cells using at leasta 1/16 dilution. See, Table 19.

TABLE 19 Stability Test Results For 2H3C5/A(6)B11MAb Combination LotManufacture Maximum Acceptable Time number date Date tested DilutionResult elapsed 0915065A Sep. 15, 2006 Sep. 19, 2006 1/256 Pass  0-months1127065A Nov. 27, 2006 Nov. 20, 2006 1/256 Pass 0523075A May 23, 2007Aug. 13, 2007 1/256 Pass 0915065A Sep. 15, 2006 Dec. 19, 2006 1/256 Pass 3-months 1127065A Nov. 27, 2006 Mar. 19, 2007 1/256 Pass 0523075A May23, 2007 Aug. 23, 2007 1/256 Pass 0915065A Sep. 15, 2006 Mar. 15, 20071/256 Pass  6-months 1127065A Nov. 27, 2006 Jun. 05, 2008 1/256 Pass0523075A May 23, 2007 Mar. 05, 2008 1/256 Pass 0915065A Sep. 15, 2006Jun. 20, 2007 1/256 Pass  9-months 1127065A Nov. 27, 2006 Aug. 28, 20071/256 Pass 0523075A May 23, 2007 Mar. 05, 2008 1/256 Pass 0915065A Sep.15, 2006 Sep. 18, 2007 1/256 Pass 12-months 1127065A Nov. 27, 2006 Dec.04, 2007 1/256 Pass 0523075A May 23, 2007 May 20, 2008 1/256 Pass0915065A Sep. 15, 2006 Dec. 18, 2007 1/256 Pass 15-months 1127065A Nov.27, 2006 Feb. 27, 2008 1/256 Pass 0523075A May 23, 2007 pending 0915065ASep. 15, 2006 Mar. 18, 2008 1/256 Pass 18-months 1127065A Nov. 27, 2006pending 0523075A May 23, 2007 pending 0915065A Sep. 15, 2006 pending24-months 1127065A Nov. 27, 2006 pending 0523075A May 23, 2007 pending

EXPERIMENTAL Example I LDFA Detection Of Influenza A Virus

Duplicate R-Mix® sv/cs cell culture monolayers were inoculated with asseries of four (4) 10-fold serial dilutions (i.e., designated assamples: 4+, 3+, 2+, and 1+) of either influenza A virus (A/H3N2) orHerpes simplex virus (HSV-1) and compared to a negative control (NC).The infected cultures were cultivated on coverslips within shell vialsto allow virus replication for approximately twenty-two (22) hours.

The culture medium was aspirated from the 4+, 1+, and NC shell vials foreach virus set. Phosphate buffered saline (PBS; 200 μl) were then addedto each shell vial; and the monolayer was scraped off of the coverslipand transferred to a labeled 1.5 ml Eppendorf centrifuge tube Acetone(100%; 800 μl) was added to each centrifuge tube to bring the finalvolume to 1 ml to create an 80% acetone/cell suspension solution Thissolution was incubated at room temperature for approximately 10 minutesto permeabilize the cells.

The permeabilized cells were then harvested and centrifuged in a Carl'smicrotube centrifuge for 6 min @ 4000 rpm. Each tube was then aspiratedto remove all liquid. Fluorescently labeled Flu A MAb or fluoresentlylabeled HSV-1 MAb (200 ul) was added to the appropriate tubes. Each cellpellet was then re-suspended in the MAb solution and incubated at 35-37°C. for 1 hour.

Subsequent to the MAb incubation, the tubes were placed back in themicro-centrifuge for 6 min @ 4000 rpm. Each tube was then aspirated toremove the MAb solution and the cells were resuspended in PBS (20 μl).An aliquot (10 μl) of each cell suspension was placed onto respectiveslides and then viewed on a widepass band FITC filter (100×magnification).

Example II Duet MAb Virus Screening In Clinical Aspirates

Nasal discharge specimens were collected from patients. An aliquot ofeach specimen was placed in an A/B tube; R/M tube; or a P/Ad tube(A—influenza A; B—influenza B; R—respiratory virus; M—metapneumovirus;P—parainfluenza virus; Ad—adenovirus).

-   -   1. Add 70 μl of cell suspension to each tube    -   2. Add 2 drops of corresponding MAb    -   3. Incubate at 35° C.-37° C. for 5 minutes    -   4. Wash with 1.5 ml of PBS    -   5. Centrifuge for 2 minutes at 2000×g    -   6. Aspirate supernatant with transfer pipette    -   7. Add 20 μl Resuspension Buffer to each tube    -   8. Load slide

Example III MAb Cross Reactivity: Listing Of Materials

The following is a listing of the materials, with lot numbers, used inthe described the cross-reactivity studies presented herein inaccordance with Examples IV and V.

Material Lot Numbers R-Mix Cultures (A549 and Mv1Lu cells) 960309 MRC-5Shell Vials C510328A H & V Mix Cultures (MRC-5 and CV-1 cells) 980309RM-03T Refeed Medium 011206A RM02 Refeed Medium 110905A 010306A HSV-1DFA control stain 013105 HSV-2 DFA control stain 013105A Influenza A DFAcontrol stain 061505A Influenza B DFA control stain 060205B AdenovirusDFA control stain 041205D Parainfluenza 1 DFA control stain 081205P1Parainfluenza 2 DFA control stain 040505P2 Parainfluenza 3 DFA controlstain 052705P3 RSV DFA control stain 052505R CMV IFA control stain031804 Chemicon VZV DFA control stain 24100183CE Adenovirus ATCC VR-1Type 1 061704J ATCC VR-3 Type 3 112701A ATCC VR-5 Type 5 070505 ATCCVR-1083 Type 6 111201A ATCC VR-7 Type 7 112701C ATCC VR-1087 Type 10111201B ATCC VR-14 Type 13 112701E ATCC VR-15 Type 14 033104 ATCC VR-19Type 18 011702A ATCC VR-1109 Type 31 011702B ATCC VR-931 Type 40 012802ATCC VR-930 Type 41 012802A Influenza A Virus ATCC VR-547 A2/Aichi/2/68strain 061704O ATCC VR-98 A/MaI/302/54 strain 061704D ATCC VR-544 A/HongKong/8/68 strain 040104 ATCC VR-546 A1/Denver/1/57 strain 061704P ATCCVR-810 A/Port Chalmers/1/73 strain 061704C ATCC VR-822 A/Victoria/3/75strain 080204 ATCC VR-897 A/New Jersey/8/76 strain 110404 ATCC VR-1520A/WS/33 strain 061704B ATCC VR-1469 A/PR/8/34 strain 061704Q Influenza BVirus ATCC VR-790 B/Russia/69 strain 041105 ATCC VR-295 B/Taiwan/2/62strain 061704E ATCC VR-103 B/GL/1739/54 strain 061704F ATCC VR-523B/Mass/3/66 strain 093004A ATCC VR-296 B/Maryland/1/59 strain 041105ATCC VR-823 B/Hong Kong/5/72 strain 093004B JH-001 Isolate, Cell CultureAdapted 061704R RSV ATCC VR-1401 RSV B Wash/18537/′62 strain 042204WATCC VR-26 Long strain 042204L ATCC VR-955 9320 strain 061704IParainfluenza 1 Virus ATCC VR-94 C-35 strain 061704L Parainfluenza 2Virus ATCC VR-92 Greer strain 061704M Parainfluenza 3 Virus ATCC VR-243C 243 strain 061704N Parainfluenza 4 Virus ATCC VR-1378 M-25 strain112701U ATCC VR-1377 CH 19503 strain 112701V Metapneumovirus Subgroup A1110905 Subgroup A2 110805 Subgroup B1 111105 Subgroup B2 110405Coronavirus ATCC VR-740 229E strain 121903 ATCC VR-1558 OC43 strain041204B Rhinovirus 39 ATCC VR-340 209 Picornavirus strain 112701EE HSV-1ATCC VR-733 F (1) strain 052405 ATCC VR-539 MacIntyre strain 071005HSV-2 ATCC VR-540 MS strain 112701Y ATCC VR-734 G strain 052605 CMV ATCCVR-977 Towne strain 011503 ATCC VR-807 Davis strain 062005 ATCC VR-538Ad-169 strain 052705 VZV ATCC VR-916 Webster strain 040504 ATCC VR-1367Ellen strain 050903 Echovirus Bion Enterprises Echovirus Panel AntigenQEC-0008 Control Slide with Echo 4, 6, 9, 11, 30, and 34. CoxsackieVirus Bion Enterprises Coxsackie Group B Antigen QCB-0011 Control Slidewith Coxsackie B1, B2, B3, B4, B5, and B6. Mumps Virus Bion EnterprisesMumps Antigen Control Slide QMU-0308 Rubeola (Measles) Virus BionEnterprises Rubeola Antigen Control Slide QME-0424 Cell Lines Tested forCross Reactivity RD (Human Rhabdomyosarcoma) C760908 Mv1Lu (Mink Lung)C580915 LLC-MK2 (Rhesus Monkey Kidney) C860928S MRHF (Human ForeskinFibroblast) C440912 NCI-H292 (Human Pulmonary Muco-Epidermoid C590929Carcinoma) BGMK (Buffalo Green Monkey Kidney) C530914 MDCK (Madin-DarbyCanine Kidney) C830921S pRHMK (Primary Rhesus Monkey Kidney) CA490922pRHMK II (pRHMK less than 3 years old) A490909YS MRC-5 (Human EmbryonicLung Fibroblast) C510920 HEp-2 (Human Epidermoid Carcinoma) C570914 pRK(Primary Rabbit Kidney) 480909 pCMK (Primary Cynomolgus Monkey Kidney)A470907 A549 (Human Lung Carcinoma) C560921 R-Mix (Mv1Lu and A549 mixedcells) C960922 WI-38 (Human Embryonic Lung Fibroblasts) 850913 Vero(African Green Monkey Kidney) C840914S Other Microorganisms/Growth MediaATCC 15531 Mycoplasma pneumoniae 031404 ATCC 23114 Mycoplasma hominis031404 ATCC 23714 Mycoplasma orale 031404 ATCC 23064 Mycoplasmasalivarium 031404 ATCC 27618 Ureaplasma urealyticum 031404 ATCC 23206Acholeplasma laidlawii 031404 ATCC 10580 Bordetella bronchiseptica031404 ATCC 10380 Bordetella pertussis 031404 ATCC 33152 Legionellapneumophila 031404 ATCC 8176 Moraxella cartarrhalis 031404 ATCC 19409Corynebacterium diphtheriae 031404 ATCC 9006 Haemophilis influenzae TypeA 031404 ATCC 33495 Klebsiella pneumoniae 031404 ATCC 9027 Pseudomonasaeruginosa 031404 ATCC 10813 Streptococcus pneumoniae 031404 ATCC 9898Streptococcus pyogenes 031404 Trichomonas vaginalis slide 25030319CEChlamydia psittaci slide FP-12-050218 Chlamydia pneumoniae slide CP-0176Chlamydia trachomatis slide 052705 Gardnerella vaginalis 410721Salmonella minnesota (enteriditis) 3457511 Neisseria gonorrhoeae 060805Salmonella typhimurium 363162 Acinetobacter calcoaceticus 934332 Candidaglabrata 992206 Escherichia coli 335472 Proteus mirabilis 440498Streptococcus agalactiae 370784 Staphylococcus aureus 081100 BG Sulfaagar Hardy Diagnostics G87 Blood agar Hardy Diagnostics 5257771 RTFCasman Agar Hardy Diagnostics A68 MacConkey agar Hardy Diagnostics G35Nickerson's Agar Hardy Diagnostics G17 Trypticase Soy Agar BD BBL 292396

Example IV Viral Cross Reactivity Protocols

A. Respiratory Viruses

1. Preparation of Frozen Stocks:

-   -   a. Influenza A and B

Amplify Influenza in MDCK T-75 cm² flasks from the original ATCCcultures as follows:

-   -   Thaw and, if necessary, add sterile water to each lyophilized        vial of Influenza and vortex for 5 to 10 seconds.    -   Remove 0.250-mL and add to 10-mL of RM03T Refeed Medium (DHI        catalog number 10-330500). Vortex for 5 to 10 seconds.    -   Aspirate medium from the flask. Rinse the flask using 10-mL        RM03T and add the 10-mL of diluted virus from step 2.    -   Place into a humidified 35° to 37° C. incubator with 5% CO₂ for        2 hours. Rock the flask every 10 to 15 minutes.    -   Add 20-mL of RM03T to the flask and return it to the incubator.    -   Monitor daily for cytopathic effect (CPE). When monolayer        reaches ˜80% to 100% CPE, place flask in a −80° C. freezer for        at least 24 hours.    -   Rapidly thaw flask in a 35° to 37° C. water bath.    -   Transfer virus-infected medium from the flask to 50-mL        polypropylene conical centrifuge tubes and vortex.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into labeled 1-mL        cryo-vials and freeze at −80° C. or lower.    -   b. Respiratory Syncytial Virus (RSV), and Parainfluenza 1, 2,        and 4

Amplify RSV in HEp-2 T-75 cm² flasks from the original ATCC cultures asfollows:

-   -   Thaw and, if necessary, add sterile water to each lyophilized        vial of RSV and vortex for 5 to 10 seconds.    -   Remove 0.250-mL and add to 10-mL of RM03T Refeed Medium (DHI        catalog number 10-330500). Vortex for 5 to 10 seconds.    -   Aspirate medium from the flask. Rinse the flask using 10-mL        RM03T and add the 10-mL of diluted virus from step 2.    -   Place into a humidified 35° to 37° C. incubator with 5% CO₂ for        2 hours. Rock the flask every 10 to 15 minutes.    -   Add 20-mL of RM03T to the flask and return it to the incubator.    -   Monitor daily for CPE. When monolayer reaches ˜80% to 100% CPE,        place flask in a −80° C. freezer for at least 24 hours.    -   Rapidly thaw flask in a 35° to 37° C. water bath.    -   Transfer virus-infected medium from the flask to 50-mL        polypropylene conical centrifuge tubes and vortex.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into labeled 1-mL        cryo-vials and freeze at −80° C. or lower.    -   c. Adenovirus

Amplify Adenovirus in A549 T-75 cm² flasks from the original ATCCcultures as follows:

-   -   Thaw and, if necessary, add sterile water to each lyophilized        vial of Adenovirus and vortex 5 to 10 seconds.    -   Remove 0.25-mL and add to 10-mL of RM03T Refeed Medium. Vortex 5        to 10 seconds.    -   Aspirate medium from flask and add the 10-mL of diluted virus        from step 2.    -   Place in a humidified 35° to 37° C. incubator with 5% CO₂ for 2        hours. Rock every 10 to 15 minutes.    -   Add 20-mL of RM03T Refeed Medium to the flask and return it to        the incubator.    -   Monitor daily for CPE. When monolayer reaches 80% to 100% CPE,        place flask in a −80° C. freezer for at least 24 hours.    -   Rapidly thaw flask in a 35° to 37° C. water bath.    -   Transfer virus-infected medium from the flask to 50-mL        polypropylene conical centrifuge tubes and vortex.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into 1-mL cryo-vials and        freeze at −80° C. or lower.

2. Determination of Respiratory Virus Concentrations

After the respiratory virus stocks are frozen, they are quantified(titered) on R-Mix cell cultures. Each virus is titered using thefollowing method:

-   -   Rapidly thaw 1 vial of appropriate virus in a 35° to 37° C. C        water bath or heating block.    -   Vortex vial and remove 0.5-mL and transfer to 4.5-mL of RM03T        Refeed Medium for a 1:10 dilution.    -   Continue making 1:10 serial dilutions to yield 1:100, 1:1,000,        1:10,000, 1:100,000, and 1:1,000,000 dilutions.    -   Aspirate culture medium from R-Mix cultures and add 1-mL of each        dilution to duplicate monolayers.    -   Centrifuge at 700×g for 60 minutes.    -   Place monolayer in a 35° to 37° C. incubator for 24 hours.    -   Aspirate medium and fix cells in 80% acetone for 5-10 minutes.        Remove acetone and add Phosphate Buffered Saline (PBS) Wash        Solution (DHI catalog number 01-001025) to prevent monolayers        from drying out.    -   Stain with specific MAb reagents and examine for fluorescence.    -   Count fluorescent foci and note the dilution counted. Calculate        the titer as follows: average count×the reciprocal of the        dilution factor=virus/mL.

These stocks may be cultured and sub-cultured on a routine basis.

Example: 250 fluorescent foci counted at a 1:10,000 dilution in a 1 mLinoculum would yield (250 foci with a 1 mL inoculum×10,000=2.5e6virus/mL). This is converted to TCID₅₀ by dividing the foci per mL by0.7 as stated by the ATCC.atcc.org/common/technicalInfo/faqAnimalVirology.cfm

3. Cross-Reactivity Testing

The R-Mix cell line containing both MvlLu and A549 cells is used forvirus isolation staining of Influenza A, Influenza B, RSV, ParainfluenzaVirus Types 1, 2, 3, 4a, 4b, and Adenovirus. Monolayers in 96-wellmicro-titer plates are used and processed according to the followingprocedure:

-   -   Rapidly thaw 1 vial of appropriate virus in a 35° to 37° C.        water bath or heating block.    -   Vortex freezer vial then dilute each Respiratory virus strain in        RM03T Refeed Medium at a 1,400 TCID₅₀ per 0.5-mL inoculum.    -   Aspirate culture medium from each 48-well plate and add 0.5 mL        of inoculum.    -   Centrifuge at 700×g for 60 minutes.    -   Place plates in a 35° to 37° C. incubator for 24 hours.    -   Remove from incubator, aspirate medium and rinse with 1-mL of        PBS.    -   Aspirate PBS then fix monolayers with 1-mL of 80% acetone for 5        minutes. Aspirate then add 0.5-mL of PBS.    -   Remove PBS and add 0.15-mL of the CMV test reagent to duplicate        monolayers. Also add 0.15-mL of the Influenza A, Influenza B,        Parainfluenza, RSV, and Adenovirus Positive Control Reagents        (DHI catalog numbers 01-013102, 01-013202, 01-013302, 01-013402,        01-013502, 01-013602, and 01-013702, and Light Diagnostics        Parainfluenza 4 reagent catalog number 5034) in duplicate        monolayers.    -   Place cultures in a 35° to 37° C. incubator for 30 minutes.    -   Rinse 2 to 3 times with 1×PBS Solution. Remove each coverslip        using a bent tip needle and place on to a drop of Mounting Fluid        cell-side down.    -   Examine for fluorescence at 100-200× total magnification and        note wells where fluorescent staining cells or background        staining is visible. Only the specific positive control reagents        should exhibit fluorescence; there should be no fluorescence        from the test MAb Reagent.

B. Herpes Simplex Virus (HSV) 1 and 2 and Cytomegalovirus (CMV)

1. Preparation of Frozen Stocks:

Amplify HSV and CMV in MRC-5 T-75 cm² flasks from the original ATCCcultures as follows:

-   -   Thaw and, if necessary, add sterile water to each lyophilized        vial of HSV or CMV and vortex 5 to 10 seconds.    -   Remove 0.250-mL and add to 10-mL of SR120 Refeed Medium (DHI        catalog number 10-200100). Vortex 5 to 10 seconds.    -   Aspirate medium from the flask. Rinse the flask using 10-mL RM02        Refeed Medium and add the 10-mL of diluted virus from step 2.    -   Place into a humidified 35° to 37° C. incubator with 5% CO₂ for        2 hours. Rock the flask every 10 to 15 minutes.    -   Add 20 mL of SR120 Refeed Medium to the flask and return it to        the incubator.    -   Monitor daily for CPE. When the monolayer reaches ˜80% to 100%        CPE, process the HSV and CMV as follows:

For HSV only:

-   -   Place HSV-infected flask in a −80° C. freezer for at least 24        hours.    -   Rapidly thaw flask in a 35° to 37° C. water bath.

For CMV only:

-   -   Scrape cells into medium in the flask.    -   Transfer to a syringe and pressure lyse through a 26-gauge        syringe needle into a 50-mL centrifuge tube.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into labeled 1-mL        cryo-vials and freeze at −80° C. or lower.

2. Determination of HSV/CMV Concentrations

The stocks are titered by the following procedure:

-   -   One vial of each strain of HSV/CMV is thawed in a 35° to 37° C.        water block.    -   Vortex each vial and remove and transfer 0.5-mL to 4.5-mL of        ELVIS® Replacement Medium (DHI catalog number 10-220100) for HSV        and RM-02 Refeed Medium (DHI catalog number 10-320100) for CMV        for a 1:10 dilution.    -   Continue making 1:10 serial dilutions to yield: 1:100, 1:1,000,        1:10,000, 1:100,000, and 1:1,000,000 dilutions.    -   Aspirate the culture medium from the ELVIS®/MRC-5 cultures and        add 1-mL of each dilution to duplicate monolayers.    -   Centrifuge at 700×g for 60 minutes.    -   Place cultures in a 35° to 37° C. incubator for 24 hours.    -   Aspirate medium and fix cells in 80% acetone for 5-10 minutes.        Remove acetone and add PBS to prevent monolayers from drying        out.    -   Stain HSV with the ELVIS Typing System and CMV with an antibody        specific to the Immediate Early CMV Antigen and examine for        fluorescence.    -   Count fluorescent foci for both CMV and HSV and note the        dilution counted. Calculate the titer as follows: average        count×the reciprocal of the dilution factor=virus/mL.

These stocks may be cultured and sub-cultured on a routine basis.

Example: 250 fluorescent foci counted at a 1:10,000 dilution in a 1 mLinoculum would yield (250 foci with a 1 mL inoculum×10,000=2.5e6virus/mL). This is converted to TCID₅₀ by dividing the foci per mL by0.7 as stated by the ATCC.

atcc.org/common/technicalInfo/faqAnimalVirology.cfm

3. Cross-Reactivity Testing

For the cross-reactivity studies, HSV and CMV strains are inoculatedinto H&V Mix (MRC-5+CV1 mix) shell vial cultures:

-   -   Each virus stock is rapidly thawed in a 35° to 37° C. water        block.    -   Dilute each HSV and CMV virus stock to be tested at a 140 TCID₅₀        per 1-mL inoculum in RM02 Refeed Medium.    -   Aspirate culture medium from each H&V Mix shell vial and add        1-mL of inoculum.    -   Centrifuge at 700×g for 60 minutes.    -   Place monolayer in a 35° to 37° C. incubator for 24 hours.    -   Remove from incubator, aspirate inoculum and rinse with 1-mL of        PBS.    -   Aspirate PBS then fix monolayers with 1-mL of acetone for 5        minutes. Aspirate then add 1-mL of PBS.    -   Remove PBS and add 0.2-mL of the CMV MAb test reagent to        duplicate shell vials of both HSV and CMV infected monolayers.        For the HSV infected monolayers, add 0.2 mL of the HSV-1 and        HSV-2 Positive Control Reagents (DHI catalog numbers 03-09510        and 03-09520) to each in duplicate. For the CMV infected shell        vials, add 0.2 mL of the CMV IE Ag Positive Control Reagent (DHI        catalog number 03-07300) to duplicate shell vials.    -   Place cultures in a 35° to 37° C. incubator for 30 minutes.    -   For the shell vials staining with the CMV IE Ag Positive        Control, rinse each vial 2 to 3 times then add 0.2-mL of CMV IE        Ag Goat Anti-Mouse Reagent (DHI catalog number 03-07400) and        incubate again for 30 more minutes.    -   Rinse 2 to 3 times with PBS. Remove each coverslip using a bent        tip needle and place on to a drop of Mounting Fluid cell-side        down.    -   Examine for fluorescence at 100-200× total magnification and        note wells where fluorescent staining cells or background        staining is visible. Only the specific positive control reagents        should exhibit fluorescence; there should be no fluorescence        from the CMV MAb test reagent on HSV-1 and HSV-2 infected        monolayers. For the CMV infected monolayers, the CMV MAb test        reagent and positive control should both show fluorescence.

C. Varicella-Zoster Virus (VZV)

1. Preparation of Frozen Stocks:

Amplify VZV in a CV-1 T-75 cm² flask from the original ATCC culture asfollows:

-   -   Thaw VZV and vortex 5 to 10 seconds.    -   Remove 0.250-mL and add to 10-mL SR120 Refeed Medium (DHI        catalog number 10-200100). Vortex 5 to 10 seconds.    -   Aspirate medium from the flask. Rinse the flask using 10-mL RM02        Refeed Medium and add the 10-mL of diluted virus from step 2.    -   Place into a humidified 35° to 37° C. incubator with 5% CO₂ for        2 hours. Rock the flask every 10 to 15 minutes.    -   Add 20-mL of SR120 Refeed Medium to the flask and return it to        the incubator.    -   Monitor daily for CPE. When the monolayer reaches ˜50% CPE,        trypsinize cells and transfer to a CV-1 T-225 cm² flask and        monitor daily for CPE. When the monolayer reaches 80% to 100%        CPE, scrape cells and pressure lyse through a 26-gauge syringe        needle into 50-mL centrifuge tubes.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into labeled 1-mL        cryo-vials and freeze at −80° C. or lower.

2. Determination of VZV Concentrations

The stocks are titered in the following manner on MRC-5 monolayers:

-   -   Rapidly thaw one vial of each VZV strain in a 35° to 37° C.        water block.    -   Vortex each vial and transfer 0.5-mL into 4.5-mL of RM-02 for a        1:10 dilution.    -   Continue making 1:10 serial dilutions to yield: 1:100, 1:1,000,        1:10,000, 1:100,000, and 1:1,000,000 dilutions.    -   Aspirate culture medium from MRC-5 monolayers and add 1-mL of        each dilution to duplicate monolayers.    -   Centrifuge at 700×g for 60 minutes.    -   Place monolayer in a 35° to 37° C. incubator for 72 hours.    -   Aspirate medium and fix cells in 80% acetone for 5-10 minutes.        Remove acetone and add PBS to prevent monolayers from drying        out.    -   Stain with a MAb specific for VZV and examine for fluorescence.    -   Count fluorescent foci and note the dilution counted. Calculate        the titer as follows: average count×the reciprocal of the        dilution factor=virus/mL.

These stocks may be used and sub-cultured on a routine basis.

Example: 250 fluorescent foci counted at a 1:10,000 dilution in a 1-mLinoculum would yield (250 foci with a 1-mL inoculum×10,000=2.5e6virus/mL). This is converted to TCID₅₀ by dividing the foci per mL by0.7 as stated by the ATCC.atcc.org/common/technicalInfo/faqAnimalVirology.cfm 3. Cross-ReactivityTesting:

For cross-reactivity studies, the VZV strains are inoculated into H&VMix (MRC-5+CV-1 mix) shell vial cultures:

-   -   Each virus stock is rapidly thawed in a 35° to 37° C. water        block.    -   Dilute each VZV strain in RM-02 Refeed Medium to yield a 140        TCID₅₀ per 1-mL inoculum.    -   Aspirate culture medium from each H&V Mix multiwell plate and        add 0.2-mL of inoculum.    -   Centrifuge at 700×g for 60 minutes.    -   Place plates in a 35° to 37° C. incubator for 72 hours.    -   Remove from incubator, aspirate inoculum and rinse with 0.2-mL        of PBS.    -   Aspirate PBS then fix monolayers with 0.2-mL of 80% acetone for        5 minutes. Aspirate then add 0.2-mL of PBS.    -   Aspirate PBS then add 0.05-mL of the subject reagent to        duplicate shell vials. Add 0.2-mL of the VZV Positive Control        Reagent (DHI Catalog number 01-025005) to duplicate wells.    -   Place cultures in a 35° to 37° C. incubator for 30 minutes.    -   Rinse 2 to 3 times with PBS. Add one drop of Mounting Fluid to        each well.    -   Examine for fluorescence at 100-200× total magnification and        note wells where fluorescent staining cells or background        staining is visible. Only the specific positive control reagents        should exhibit fluorescence; there should be no fluorescence        from the test MAbs.

D. Rhinovirus 39

1. Preparation of Frozen Stocks:

Amplify Rhinovirus in a MRC-5 T-75 cm² flask from the original ATCCculture as follows:

-   -   Thaw Rhinovirus and vortex 5 to 10 seconds.    -   Remove 0.250-mL and add to 10-mL SR120 Refeed Medium (DHI        catalog number 10-200100). Vortex 5 to 10 seconds.    -   Aspirate medium from the flask. Rinse the flask using 10-mL RM02        Refeed Medium and add the 10-mL of diluted virus from step b.    -   Place into a humidified 33°-35° C. incubator with 5% CO₂ for 2        hours. Rock the flask every 10 to 15 minutes.    -   Add 20-mL of SR120 Refeed Medium to the flask and return it to        the incubator.    -   Monitor daily for CPE (cytopathic effect). When the monolayer        reaches ˜80-100% CPE, freeze flask in a −80° C. freezer for at        least 24 hours.    -   Thaw flask in a 35°-37° C. water bath until just thawed.    -   Transfer virus-infected medium from the flask to 50-mL        polypropylene conical centrifuge tubes and vortex.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into labeled 1-mL        cryo-vials and freeze at −80° C. or lower.

2. Determination of Rhinovirus Concentrations:

The stocks are titered in the following manner on MRC-5 monolayers:

-   -   Rapidly thaw one vial of Rhinovirus 39 in a 35°-37° C. water        block.    -   Vortex each vial and transfer 0.5-mL into 4.5-mL of RM-02 for a        1:10 dilution.    -   Continue making 1:10 serial dilutions to yield: 1:100, 1:1,000,        1:10,000, 1:100,000, and 1:1,000,000 dilutions.    -   Aspirate culture medium from MRC-5 monolayers and add 1-mL of        each dilution to duplicate monolayers.    -   Centrifuge at 700×g for 60 minutes.    -   Place monolayer in a 33°-35° C. incubator until CPE is observed.        Note Day and dilution.        These stocks may be cultured and sub-cultured on a routine        basis.

3. Cross Reactivity Testing:

For cross reactivity studies, the Rhinovirus is inoculated in to MRC-5cell cultures:

-   -   Thaw virus stock rapidly in a 35°-37° C. water block.    -   Dilute the Rhinovirus in RM-02 Refeed Medium to yield a 1,400        TCID₅₀ per 1-mL inoculum.    -   Aspirate culture medium from each MRC-5 shell vial and add 1-mL        of inoculum.    -   Centrifuge at 700×g for 60 minutes.    -   Place plates in a 33°-35° C. incubator for 24 hours.    -   Remove from incubator, aspirate inoculum and rinse with 1-mL of        PBS.    -   Aspirate PBS then fix monolayers with 1-mL of 80% acetone for 5        minutes. Aspirate then add 1-mL of PBS.    -   Aspirate PBS then add 0.2-mL of the subject reagent to duplicate        wells.    -   Place cultures in a 35°-37° C. incubator for 30 minutes    -   Rinse 2 to 3 times with PBS. Remove coverslip using a bent-tip        needle and place, cell-side down, on to one drop of Mounting        Fluid.    -   Examine for fluorescence at 100× total magnification. The viral        plaques should not exhibit any fluorescent staining nor should        there be an excess of background staining

E. Coronaviruses

1. Preparation of Frozen Stocks:

Amplify Coronaviruses in MRC-5 T-75 cm² flasks from the original ATCCcultures as follows:

-   -   Thaw Coronaviruses and vortex 5 to 10 seconds.    -   Remove 0.250-mL and add to 10-mL SR120 Refeed Medium (DHI        catalog number 10-200100). Vortex 5 to 10 seconds.    -   Aspirate medium from the flask. Rinse the flask using 10-mL RM02        Refeed Medium and add the 10-mL of diluted virus from step b.    -   Place into a humidified 33°-35° C. incubator with 5% CO₂ for 2        hours. Rock the flask every 10 to 15 minutes.    -   Add 20-mL of SR120 Refeed Medium to the flask and return it to        the incubator.    -   Monitor daily for CPE (cytopathic effect). When the monolayer        reaches ˜80-100% CPE, freeze flask in a −80° C. freezer for at        least 24 hours.    -   Thaw flask in a 35°-37° C. water bath until just thawed.    -   Transfer virus infected-medium from the flask to 50-mL        polypropylene conical centrifuge tubes and vortex.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into labeled 1-mL        cryo-vials and freeze at −80° C. or lower.

2. Determination of Coronavirus Concentrations:

The Coronavirus stocks are titered in the following manner on MRC-5monolayers:

-   -   Rapidly thaw one vial of each Coronavirus in a 35°-37° C. water        block.    -   Vortex each vial and transfer 0.5-mL into 4.5-mL of RM-02 for a        1:10 dilution.    -   Continue making 1:10 serial dilutions to yield: 1:100, 1:1,000,        1:10,000, 1:100,000, and 1:1,000,000 dilutions.    -   Aspirate culture medium from MRC-5 monolayers and add 1-mL of        each dilution to duplicate monolayers.    -   Centrifuge at 700×g for 60 minutes.    -   Place monolayers in a 33°-35° C. incubator for 16-24 hours.    -   Aspirate medium and fix cells in 80% acetone for 5-10 minutes.        Remove acetone and add PBS to prevent monolayers from drying        out.    -   Stain with a research use only monoclonal antibody for        Coronaviruses and examine for fluorescence.    -   Count fluorescent foci and note the dilution counted. Calculate        the titer as follows: average count×the reciprocal of the        dilution factor=virus/mL.        These stocks may be cultured and subcultured on a routine basis.        Example: 250 fluorescent foci counted at a 1:10,000 dilution in        a 1-mL inoculum would yield (250 foci with a 1-mL        inoculum×10,000=2.5e6 virus/mL). This is converted to TCID₅₀ by        dividing the foci per mL by 0.7 as stated by the ATCC.        atcc.org/common/technicalInfo/faqAnimalVirology.cfm

3. Cross Reactivity Testing

For cross reactivity studies, the Coronaviruses are inoculated in toMRC-5 cell cultures:

-   -   Thaw virus stock rapidly in a 35°-37° C. water block.    -   Dilute the Coronaviruses in RM-02 Refeed Medium to yield a 1,400        TCID₅₀ per 1-mL inoculum.    -   Aspirate culture medium from each MRC-5 shell vial and add 1-mL        of inoculum.    -   Centrifuge at 700×g for 60 minutes.    -   Place plates in a 33°-35° C. incubator for 24 hours.    -   Remove from incubator, aspirate inoculum and rinse with 1-mL of        PBS.    -   Aspirate PBS then fix monolayers with 1-mL of 80% acetone for 5        minutes. Aspirate then add 1-mL of PBS.    -   Aspirate PBS then add 0.2-mL of the subject reagent to duplicate        wells. Add 0.2-mL of the research use only monoclonal antibody        reagent in duplicate.    -   Place cultures in a 35°-37° C. incubator for 30 minutes    -   Rinse 2 to 3 times with PBS. Remove coverslip using a bent-tip        needle and place, cell-side down, on to one drop of Mounting        Fluid.    -   Examine for fluorescence at 100× total magnification. The        subject reagent should not exhibit any fluorescent staining or        excessive background staining. The positive controls should        exhibit bright apple-green fluorescence.

F. Metapneumovirus

1. Preparation of Frozen Stocks

Amplify Metapneumovirus (MPV) subgroups in LLC-MK2 T-75 cm² flasks fromstocks obtained from the University of Pavia, Italy:

-   -   Thaw all MPV subgroup vials and vortex 5 to 10 seconds.    -   Remove 0.250-mL and add to 10-mL SR120 Refeed Medium (DHI        catalog number 10-200100). Vortex 5 to 10 seconds.    -   Aspirate medium from the flask. Rinse the flask using 10-mL RM02        Refeed Medium and add the 10-mL of diluted virus from step 2.    -   Place into a humidified 35°-37° C. incubator with 5% CO₂ for 2        hours. Rock the flask every 10 to 15 minutes.    -   Add 20-mL of SR120 Refeed Medium to the flask and return it to        the incubator.    -   Monitor daily for CPE (cytopathic effect). When the monolayer        reaches ˜80-100% CPE, scrape cells from the flask into        suspension.    -   Pressure-lyse each virus suspension separately through a        26-gauge needle.    -   Transfer lysed virus/cell suspension to 50-mL polypropylene        conical centrifuge tubes and vortex.    -   Centrifuge at 300×g for 10 minutes to pellet cell debris.    -   Remove supernatant, taking care not to disturb pellet, transfer        to another centrifuge tube and vortex.    -   Dispense 1-mL of the virus suspension into labeled 1-mL        cryo-vials and freeze at −80° C. or lower.

2. Determination of Metapneumovirus Concentrations

The stocks are titered in the following manner on R-Mix monolayers:

-   -   Rapidly thaw one vial of each MPV subgroup in a 35°-37° C. water        block.    -   Vortex each vial and transfer 0.5-mL into 4.5-mL of RM-03T for a        1:10 dilution.    -   Continue making 1:10 serial dilutions to yield: 1:100, 1:1,000,        1:10,000, 1:100,000, and 1:1,000,000 dilutions.    -   Aspirate culture medium from R-Mix monolayers and add 1-mL of        each dilution to duplicate monolayers.    -   Centrifuge at 700×g for 60 minutes.    -   Place monolayers in a 35°-37° C. incubator for 24 hours.    -   Aspirate medium and fix cells in 80% acetone for 5-10 minutes.        Remove acetone and add PBS Wash Solution (DHI catalog number        01-001025) to prevent monolayers from drying out.    -   Stain with MPV monoclonal antibody reagent and examine for        fluorescence.    -   Count fluorescent foci and note the dilution counted. Calculate        the titer as follows: average count×the reciprocal of the        dilution factor=virus/mL.

These stocks may be cultured and subcultured on a routine basis.

Example: 250 fluorescent foci counted at a 1:10,000 dilution in a 1-mLinoculum would yield (250 foci with a 1-mL inoculum×10,000=2.5e6virus/mL). This is converted to TCID₅₀ by dividing the foci per mL by0.7 as stated by the ATCC.atcc.org/common/technicalInfo/faqAnimalVirology.cfm

3. Cross Reactivity Testing:

For cross reactivity studies, the MPV subgroups are inoculated in toR-Mix cell cultures:

-   -   Rapidly thaw 1 vial of appropriate virus in a 35°-37° C. water        bath or heating block.    -   Vortex freezer vial then dilute each MPV subgroup in RM03T        Refeed Medium at a 1,400 TCID₅₀ per 0.2-mL inoculum.    -   Aspirate culture medium from each 96-well plate and add 0.2-mL        of inoculum.    -   Centrifuge at 700×g for 60 minutes.    -   Place monolayer in a 35°-37° C. incubator for 24 hours.    -   Remove from incubator, aspirate medium and rinse with 0.2-mL of        PBS.    -   Aspirate PBS then fix monolayers with 0.2-mL of 80% acetone for        5 minutes. Aspirate then add 0.2-mL of PBS.    -   Remove PBS and add 0.05-mL of the subject reagent to duplicate        monolayers. Also add 0.05-mL of the DHI MPV ASR as a positive        control.    -   Place cultures in a 35°-37° C. incubator for 30 minutes.    -   Rinse 2 to 3 times with PBS Wash Solution. Add 1 drop of        Mounting Fluid to each stained well.    -   Examine for fluorescence at 100× total magnification and note        wells where fluorescent staining cells or background staining is        visible. Fluorescence should not be observed in monolayers        stained with the subject reagent. All 4 MPV subgroups should        exhibit fluorescent staining cells using the MPV positive        control reagent.

G. Echovirus, Coxsackie Virus, Measles, and Mumps

The following control slides were purchased from Bion Enterprises forthe purpose of MAb screening and cross-reactivity studies. Each slide isindividually foil-wrapped with wells containing microorganisms of tissueculture cells infected with a specific viral agent in addition to wellscontaining only the uninfected tissue culture cells. The infected tissueculture cells serve as a positive control and the uninfected tissueculture cells serve as a negative control. The specific microbialantigen is identified on the product label.

The Echovirus Panel (catalog number QEC-6506) contains six wells, eachcontaining a mix of infected and uninfected cells. Each slide iscomprised separately of Echovirus types 4, 6, 9, 11, 30, and 34.

The Coxsackie Virus Panel (catalog number QCB-2506) contains six wells,each containing a mix of infected and uninfected cells. Each slide iscomprised separately of Coxsackie Virus types B1, B2, B3, B4, B5, andB6.

The Mumps Antigen Control Slides (catalog number QMU-8002) contain onewell of Mumps infected cells and one well of uninfected cells.

The Measles Antigen Control Slides (catalog number QME-0424) contain onewell of Measles infected cells and one well of uninfected cells.

The procedure for testing and staining of the antigen control slides is:

-   -   Stain each slide in duplicate with 0.03-mL per well of the        subject test reagent.    -   Place the slides into a 35°-37° C. incubator for 30 minutes.    -   Remove from the incubator and gently rinse slides with PBS. Blot        each slide dry while trying not to touch or disturb the cell        spots.    -   Add one drop of Mounting Fluid to each well and place a        coverglass on each slide.    -   Examine for fluorescence at 100× total magnification and note        wells where fluorescent staining cells or background staining is        visible.

H. Uninfected Cell Cultures

Uninfected cell cultures in shell vial format and glass, round-bottomtubes are tested for cross reactivity by the following procedures. Table20.

TABLE 20 Cell Culture Formats Used in Cross Reactivity Studies CellLines Medium/Format RD (Human Rhabdomyosarcoma) Shell Vial Mv1Lu (MinkLung) Shell Vial LLC-MK2 (Rhesus Monkey Kidney) Shell Vial MRHF (HumanForeskin Fibroblast) Shell Vial NCI-H292 (Human PulmonaryMuco-epidermoid Shell Vial carcinoma) BGMK (Buffalo Green Monkey Kidney)Shell Vial MDCK (Madin-Darby Canine Kidney) Shell Vial pRHMK (PrimaryRhesus Monkey Kidney) Glass Round Tube pRHMK II (pRHMK less than 3 yearsold) Glass Round Tube MRC-5 (Human Embryonic Lung Fibroblast) Shell VialHEp-2 (Human Epidermoid Carcinoma) Shell Vial pRK (Primary RabbitKidney) Shell Vial pCMK (Primary Cynomolgus Monkey Kidney) Glass RoundTube A549 (Human Lung Carcinoma) Shell Vial R-Mix (Mv1Lu and A549 mixedcells) Shell Vial WI-38 (Human Embryonic Lung Fibroblasts) Glass RoundTube Vero (African Green Monkey Kidney) Shell Vial

1. Shell Vial Procedure

-   -   Aspirate culture medium and rinse once using PBS.    -   Aspirate PBS and add 1-mL of 100% acetone fixative for 10        minutes.    -   Aspirate the fixative and add 1-mL of PBS.    -   Aspirate the PBS then add 0.2-mL of the subject reagent in        duplicate then place into a 35°-37° C. incubator for 30 minutes.    -   Remove from incubator then rinse 2-3 times with PBS.    -   Using forceps and a bent-tip needle, remove each coverslip and        place on to a drop of Mounting Fluid on a glass specimen slide.    -   Examine for fluorescence at 100× total magnification and note if        fluorescent staining cells or background staining is visible.

2. Glass Round-Bottom Tube Procedure:

-   -   Use a 2-mL serological pipette and scrape the cell monolayer        into the culture medium.    -   Transfer to 1.5-mL Eppendorf tube and centrifuge at 300×g for 10        minutes.    -   Aspirate supernatant and resuspend pellet in 1-mL of PBS.    -   Spot 8-well specimen slides with 0.01-mL. Allow to air dry for        ˜20 minutes.    -   Fix the specimen slides for 10 minutes in 100% acetone.    -   Add 0.03-mL of the subject reagent in duplicate wells of each        cell line.    -   Place into a 35°-37° C. incubator for 30 minutes.    -   Remove from incubator then rinse 2-3 times with PBS.    -   Add one drop of Mounting Medium to each well and examine for        fluorescence at 100× total magnification. Note if fluorescent        staining cells or background staining is visible.

Example V Bacterial Cross Reactivity Testing

A. Mycoplasma sp., Ureaplasma sp., and Acholeplasma laidlawii

1. Preparation of Frozen Stocks:

-   -   Thaw and reconstitute cultures from the ATCC.    -   Dilute cultures 1:10 and 1:100 with the broth supplied in        MYCOTRIM® TC Triphasic Culture System (Irvine Scientific catalog        number T500-000) for the detection of cultivatable mycoplasma.    -   Inoculate with 1-mL of the diluted bacteria by scoring the agar        on the top of the flask and then releasing the remaining        inoculum into the broth in the bottom of the flask. Note: An        undiluted sample is not inoculated because it may contain        preservatives that inhibit growth of the bacteria.    -   Place flasks at 35° to 37° C. Examine flasks daily for the        appearance of “fried egg” colonies in the agar and turbidity in        the medium.    -   When colonies are observed (about 6 to 7 days post-inoculation),        scrape the bottom of the flask into the broth and transfer to a        1.5-mL Eppendorf centrifuge vial. Also add 0.2-mL of Dienes        Stain to the agar side of the flask and stain for 30 minutes at        35° to 37° C. This stain will aid in viewing the colonies.    -   Centrifuge at 9,000×g in a microcentrifuge for 30 minutes.        Aspirate off supernatant and resuspend pellet in 50% glycerol        and freeze at −80° C.

2. Cross-Reactivity Testing:

Each bacterium is grown for cross-reactivity studies, prepared onslides, and concentrations concurrently verified using the followingprocedure:

-   -   Each bacteria stock vial is rapidly thawed in a 35° to 37° C.        water block.    -   Vortex each vial and transfer into a 1.5-mL Eppendorf tube.    -   Centrifuge at 9,000×g for 30 minutes.    -   Aspirate the supernatant and resuspend the pellet in 1-mL of        PBS.    -   Dilute cultures 1:100 and 1:1000 with the broth supplied in        MYCOTRIM® TC Triphasic Culture System.    -   Inoculate with 1-mL of the diluted bacteria by scoring the agar        on the top of the flask and then releasing the remaining        inoculum into the broth in the bottom of the flask.    -   Place flasks at 35° to 37° C. Examine flasks daily for the        appearance of “fried egg” colonies in the agar and turbidity in        the medium.    -   When colonies are observed, scrape the bottom of the flask into        the broth and transfer to a 1.5-mL Eppendorf centrifuge vial.        Also add 0.2-mL of Dienes Stain to the agar side of the flask        and stain for 30 minutes at 35° to 37° C. This stain will aid in        viewing the colonies.    -   Centrifuge at 9,000×g for 30 minutes.    -   Aspirate the supernatant and resuspend the pellet in 1-mL of        PBS.    -   Using a spectrophotometer, read 0.1-mL of each suspension in a        96-well microtiter plate at 600 nm. Include McFarland Turbidity        Standards (Scientific Device Laboratory catalog number 2350)        ranging from 0.5 to 4.0 on the same plate.    -   Based on the O.D. values, dilute each bacteria suspension in PBS        to closely match the McFarland standard of 2.0 equaling        approximately 6.0e6 CFU per mL.    -   Vortex suspensions then add 0.01-mL per well to 8-well glass        slides at both McFarland adjusted concentrations.    -   Let each slide air dry then fix in acetone for 10 minutes.    -   Store unused slides in an air tight pouch with a desiccant        pouch.    -   Each suspension adjusted to a McFarland Standard of 2.0 used to        make slides is diluted 1:1000, 1:1,000,000, and 100,000,000 in        broth supplied with the MYCOTRIM® TC Triphasic Culture System.    -   Inoculate with each 1-mL of the dilution series of bacteria by        scoring the agar on the top of the flask and then releasing the        remaining inoculum into the broth in the bottom of the flask.    -   Place flasks at 35° to 37° C. Examine flasks daily for the        appearance of “fried egg” colonies in the agar and turbidity in        the medium.    -   Note at which dilution series, colonies are still visible in the        flask.        Each 8-well slide is stained with the subject reagent by the        following procedure:    -   Stain duplicate wells of each bacteria slide with 30-4, per well        of the CMV MAb test reagent.    -   Place the slides into a 35° to 37° C. incubator for 60 minutes.    -   Remove from incubator then gently rinse slides with PBS. Blot        dry trying not to touch or disturb the cell spots.    -   Add a drop of Mounting Fluid to each well and place a coverglass        upon each slide.    -   Examine for fluorescence at 200 and 400× total magnification and        note wells where fluorescent staining cells or background        staining is visible.

B. Bordetella sp., Legionella p., Moraxella sp., Corynebacterium sp.,Haemophilis sp., Klebsiella sp., Pseudomonas sp., Streptococcus sp.,Neisseria gonorrhoeae, Staphylococcus aureus

1. Preparation of Frozen Stocks:

Stocks of each were obtained from the ATCC and grown on the appropriateagar listed below:

-   -   Buffered Charcoal Yeast Extract: Bordetella sp. and Legionella        pneumophilia    -   Blood Agar: Moraxella cartarrhalis, Corynebacterium diphtheriae,        and Streptococcus pneumoniae.    -   Trypticase Soy Agar: Neisseria gonorrhoeae, Klebsiella        pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus    -   Chocolate Agar: Haemophilis influenzae type A        These microorganisms are grown using the following procedure:    -   Dilute bacteria in Trypticase Soy Broth at 1:10 and 1:100        dilutions and absorb 1-mL of the suspension on the appropriate        agar.    -   Place agar plates face down in a 35° to 37° C. humidified        incubator. Check daily for colonies.    -   When colonies are observed, use a sterile 0.01-mL loop and        transfer colonies to a 1.5-mL Eppendorf microcentrifuge tube        containing 1-mL of PBS. Remove enough colonies to make the PBS        turbid.    -   Centrifuge at 9,000×g for 30 minutes. Aspirate off supernatant        and resuspend pellet in 50% glycerol and freeze at −80° C.

2. Cross-Reactivity Testing

Each bacterium is grown for cross-reactivity studies, prepared onslides, and concentrations concurrently verified using the followingprocedure:

-   -   Rapidly thaw each bacteria stock in a 35° to 37° C. water block.    -   Dilute bacteria in Trypticase Soy Broth at 1:10 and 1:100        dilutions and absorb 1-mL of the suspension on the appropriate        agar.    -   Place agar plates face down in a 35° to 37° C. humidified        incubator. Check daily for colonies.    -   When colonies are observed, use a sterile 0.01-mL loop and        transfer individual colonies to an Eppendorf microcentrifuge        tube containing 1-mL of PBS. Remove enough colonies to make the        PBS turbid.    -   Use a sterile 0.01-mL loop and streak each specimen on a        suitable agar.    -   Place agar plates face down in a 35° to 37° C. humidified        incubator. Check daily for colonies.    -   When colonies are observed, use a sterile 0.01-mL loop and        transfer colonies to a 1.5-mL Eppendorf centrifuge tube        containing 1 mL of PBS. Remove enough colonies to make the PBS        turbid.    -   Using a spectrophotometer, read 0.1-mL of each suspension in a        96-well microtiter plate at 600 nm. Include McFarland Turbidity        Standards (Scientific Device Laboratory catalog number 2350)        ranging from 0.5 to 4.0 on the same plate.    -   Based on the O.D. values, dilute each bacteria suspension in PBS        to closely match the McFarland standard of 1.0 and 2.0 equaling        approximately 3.0e6 and 6.0e6 CFU per mL.    -   Vortex suspensions then add 0.01-mL per well to 8-well glass        slides at both McFarland adjusted concentrations.    -   Let each slide air dry then fix in acetone for 10 minutes.    -   Store unused slides in an air tight pouch with a desiccant        pouch.    -   Each suspension at a McFarland Standard of 1.0 used to make        slides is diluted 1:100, 1:10,000, 1:1,000,000, 1:10,000,000 and        100,000,000. 1-mL of each dilution from each bacterium is        absorbed on to the appropriate agar plate for colony        confirmation.    -   Place agar plates face down in a 35° to 37° C. humidified        incubator. Check daily for colonies.    -   Count the colonies of the dilution plates with approximately        30-300 colonies. Multiply the count by the reciprocal of the        dilution factor to calculate the CFU per mL.        Each 8-well slide is stained with the CMV MAb test reagent by        the following procedure:    -   Stain duplicate wells of each bacteria slide with 30-μL per well        of the CMV MAb test reagent.    -   Place the slides into a 35° to 37° C. incubator for 60 minutes.    -   Remove from incubator then gently rinse slides with PBS. Blot        dry trying not to touch or disturb the cell spots.    -   Add a drop of Mounting Fluid to each well and place a coverglass        upon each slide.    -   Examine for fluorescence at 200 and 400× total magnification and        note wells where fluorescent staining cells or background        staining is visible.

C. Gardnerella vaginalis, Salmonella sp., Acinetobacter calcoaceticus,Candida Glabrata, Escherichia Coli, Proteus Mirabilis, StreptococcusAgalactiae

1. Preparation of Frozen Stocks:

Lyophilized discs of each were obtained from Hardy Diagnostics and grownon the appropriate agar:

-   -   BG Sulfa agar: Salmonella sp.    -   Blood agar: Streptococcus agalactiae    -   RTF Casman agar: Gardnerella vaginalis    -   MacConkey agar: Proteus mirabilis, Acinetobacter calcoaceticus,        Escherichia coli    -   Nickerson's agar: Candida glabrata

These microorganisms were reconstituted and grown in the followingmanner:

-   -   Remove the LYFO DISK vial from 4-8° C. storage and allow the        unopened vial to equilibrate to room temperature.    -   Aseptically remove one gelatin pellet from the vial. Place the        pellet in 0.5-mL of sterile Brain Heart Infusion Broth (Hardy        Diagnostics catalog number R10).    -   Emulsify and crush pellet with a sterile swab or pipette until        the pellet particles are uniform in size and the suspension is        homogenous in appearance.    -   Saturate the swab immediately with the hydrated material and        transfer the material to an appropriate, non-selective, nutrient        or enriched agar medium. With pressure, rotate the swab, and        inoculate a circular area (i.e., one inch or 25 mm in diameter)        of the agar medium. Using the same swab or a sterile loop,        repeatedly (about 10 to 20 times) streak through the inoculated        area and then continue to streak the remainder of the agar        surface.    -   When colonies are observed, use a sterile 0.01-mL loop and        transfer colonies to a 1.5-mL Eppendorf microcentrifuge tube        containing 1-mL of PBS. Remove enough colonies to make the PBS        turbid.    -   Centrifuge at 9,000×g for 30 minutes. Aspirate off supernatant        and resuspend pellet in 50% glycerol and freeze at −80° C.

2. Cross-Reactivity Testing

Each bacterium is grown for cross-reactivity studies, prepared onslides, and concentrations concurrently verified using the followingprocedure:

-   -   Rapidly thaw each bacteria stock in a 35° to 37° C. water block.    -   Dilute bacteria in Trypticase Soy Broth at 1:10 and 1:100        dilutions and absorb 1-mL of the suspension on the appropriate        agar.    -   Place agar plates face down in a 35° to 37° C. humidified        incubator. Check daily for colonies.    -   When colonies are observed, use a sterile 0.01-mL loop and        transfer individual colonies to an Eppendorf microcentrifuge        tube containing 1-mL of PBS. Remove enough colonies to make the        PBS turbid.    -   Use a sterile 0.01-mL loop and streak each specimen on a        suitable agar.    -   Place agar plates face down in a 35° to 37° C. humidified        incubator. Check daily for colonies.    -   When colonies are observed, use a sterile 0.01-mL loop and        transfer colonies to a 1.5-mL Eppendorf centrifuge tube        containing 1-mL of PBS. Remove enough colonies to make the PBS        turbid.    -   Using a spectrophotometer, read 0.1-mL of each suspension in a        96-well microtiter plate at 600 nm. Include McFarland Turbidity        Standards (Scientific Device Laboratory catalog number 2350)        ranging from 0.5 to 4.0 on the same plate.    -   Based on the O.D. values, dilute each bacteria suspension in PBS        to closely match the McFarland standard of 1.0 and 2.0 equaling        approximately 3.0e6 and 6.0e6 CFU per mL.    -   Vortex suspensions then add 0.01-mL per well to 8-well glass        slides at both McFarland adjusted concentrations.    -   Let each slide air dry then fix in acetone for 10 minutes.    -   Store unused slides in an air tight pouch with a desiccant        pouch.    -   Each suspension at a McFarland Standard of 1.0 used to make        slides is diluted 1:100, 1:10,000, 1:100,000, 1:1,000,000,        1:10,000,000 and 100,000,000. 1-mL of each dilution from each        bacterium is absorbed on to the appropriate agar plate for        colony confirmation.    -   Place agar plates face down in a 35° to 37° C. humidified        incubator. Check daily for colonies.    -   Count the colonies of the dilution plates with approximately        30-300 colonies. Multiply the count by the reciprocal of the        dilution factor to calculate the CFU per mL.        Each 8-well slide is stained with the CMV MAb test reagent by        the following:    -   Stain duplicate wells of each bacteria slide with 30-μL per well        of the CMV MAb test reagent.    -   Place the slides into a 35° to 37° C. incubator for 60 minutes.    -   Remove from incubator then gently rinse slides with PBS. Blot        dry trying not to touch or disturb the cell spots.    -   Add a drop of Mounting Fluid to each well and place a coverglass        upon each slide.    -   Examine for fluorescence at 200 and 400× total magnification and        note wells where fluorescent staining cells or background        staining is visible.

D. Trichomonas vaginalis, Chlamydia psittaci, Chlamydia trachomatis:

These microorganisms are fixed antigen control slides. The Trichomonas.vaginalis (catalog number 5073-5) and Chlamydia pneumoniae ControlSlides (catalog number CP-4212) were obtained from Chemicon/LightDiagnostics.

-   The Chlamydia psittaci (catalog number 210-88-12-FC) were purchased    from VMRD, Inc.-   The Chlamydia trachomatis (catalog number 01-00011) slides are    manufactured by Diagnostic Hybrids as a commercial product.    Each slide is stained with the CMV MAb test reagent by the following    procedure:    -   Stain duplicate slides of each bacteria with 30-μL per well of        the CMV MAb test reagent.    -   Place the slides into a 35° to 37° C. incubator for 60 minutes.    -   Remove from incubator then gently rinse slides with PBS. Blot        dry trying not to touch or disturb the cell spots.    -   Add a drop of Mounting Fluid to each well and place a coverglass        upon each slide.    -   Examine for fluorescence at 200 and 400× total magnification and        note wells where fluorescent staining cells or background        staining is visible.

The invention claimed is:
 1. A method for detection of an intracellularantigen comprising: incubating a suspension comprising a biologicalsample, at least two differentially fluorescently labeled antibodies anda staining reagent to generate a liquid cell suspension, wherein saidbiological sample is suspected of comprising at least one intracellularrespiratory viral antigen and wherein said liquid cell suspensioncomprises unfixed cells, wherein during the incubating, the at least oneintracellular viral antigen binds to one of the at least twodifferentially fluorescently labeled antibodies to form an intracellularantigen-antibody complex, wherein said respiratory viral antigen isselected from the group consisting of a respiratory syncytial viralantigen, an influenza A viral antigen, an influenza B viral antigen, anadenovirus viral antigen, a parainfluenza viral antigen and ametapneumovirus viral antigen; detecting the intracellularantigen-antibody complex within the liquid cell suspension byidentifying the one of the at least two differentially fluorescentlylabeled antibodies.
 2. The method of claim 1, wherein each of the atleast two differentially fluorescently labeled antibodies comprises aR-phycoerythrin fluorescent label or a fluorescein isothiocyanatefluorescent label.
 3. The method of claim 1, wherein each of the atleast two differentially fluorescently labeled antibodies is amonoclonal antibody.
 4. The method of claim 1, wherein the stainingreagent is detectable by fluorescent microscopy.
 5. The method of claim1, wherein the staining reagent is selected from the group consisting ofEvans blue, propidium iodide, acridine orange and combinations thereof.6. The method of claim 1, wherein at least one of the antibodiescomprises specific affinity for said respiratory syncytial viralantigen.
 7. The method of claim 1, wherein at least one of theantibodies comprises a specific affinity for said influenza A viralantigen.
 8. The method of claim 1, wherein at least one of theantibodies comprises a specific affinity for said influenza B viralantigen.
 9. The method of claim 1, wherein at least one of theantibodies comprises a specific affinity for said adenovirus viralantigen.
 10. The method of claim 1, wherein at least one of the labeledantibodies comprises a specific affinity for said parainfluenza viralantigen.
 11. The method of claim 1, wherein at least one of the labeledantibodies comprises a specific affinity for said metapneumovirus viralantigen.
 12. The method of claim 1, wherein said suspension furthercomprises a permeabilization agent.
 13. The method of claim 12, whereinsaid permeabilizaton agent comprises a detergent.
 14. The method ofclaim 13, wherein said detergent is sapogenin.